Compositions and methods for the modulation of viral maturation

ABSTRACT

This application describes a family of nucleic acid sequences and proteins encoded thereby that play a role in viral maturation: the Alternate Viral Maturation Scaffolding Protein, or the AVMSP family of proteins.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.60/308,958, filed Jul. 31, 2001, and 60/345,846, filed Nov. 9, 2001. Theentire contents of these applications are herein incorporated byreference.

BACKGROUND

Viral maturation requires the proteolytic processing of viral proteins,such as Gag, and the activity of the host proteins. It is believed thatcellular machineries for exo/endocytosis and for ubiquitin conjugationmay be involved in the maturation. In particular, the assembly andsubsequent budding of retroviruses, rhabdoviruses, and filovirusesdepends on the Gag polyprotein. After its synthesis, Gag is targeted tothe plasma membrane where it induces budding of nascent virus particles.

The role of ubiquitin in virus assembly was suggested by Dunigan et al.(1988, Virology 165, 310, Meyers et al. 1991, Virology 180, 602), whoobserved that mature virus particles were enriched in unconjugatedubiquitin. More recently, it was shown that proteasome inhibitorssuppress the release of HIV-1, HV-2 and virus-like particles derivedfrom SIV and RSV Gag. Also, inhibitors affect Gag processing andmaturation into infectious particles (Schubert et al 2000, PNAS 97,13057, Harty et al. 2000, PNAS 97, 13871, Strack et al. 2000, PNAS 97,13063, Patnaik et al. 2000, PNAS 97, 13069).

It is well known in the art that ubiquitin-mediated proteolysis is themajor pathway for the selective, controlled degradation of intracellularproteins in eukaryotic cells. Ubiquitin modification of a variety ofprotein targets within the cell appears to be important in a number ofbasic cellular functions such as regulation of gene expression,regulation of the cell-cycle, modification of cell surface receptors,biogenesis of ribosomes, and DNA repair. One major function of theubiquitin-mediated system is to control the half-lives of cellularproteins. The half-life of different proteins can range from a fewminutes to several days, and can vary considerably depending on thecell-type, nutritional and environmental conditions, as well as thestage of the cell-cycle.

Targeted proteins undergoing selective degradation, presumably throughthe actions of a ubiquitin-dependent proteosome, are covalently taggedwith ubiquitin through the formation of an isopeptide bond between theC-terminal glycyl residue of ubiquitin and a specific lysyl residue inthe substrate protein. This process is catalyzed by aubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme(E2), and in some instances may also require auxiliary substraterecognition proteins (E3s). Following the linkage of the first ubiquitinchain, additional molecules of ubiquitin may be attached to lysine sidechains of the previously conjugated moiety to form branchedmulti-ubiquitin chains.

The conjugation of ubiquitin to protein substrates is a multi-stepprocess. In an initial ATP requiring step, a thioester is formed betweenthe C-terminus of ubiquitin and an internal cysteine residue of an E1enzyme. Activated ubiquitin is then transferred to a specific cysteineon one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitinto protein substrates. Substrates are recognized either directly byubiquitin-conjugated enzymes or by associated substrate recognitionproteins, the E3 proteins, also known as ubiquitin ligases.

SUMMARY

It is proposed that a variety of proteins, including ubiquitin proteinligases and proteins involved in membrane trafficking, are recruited forthe process of viral maturation (including, for example, assembly,budding and release) by direct or indirect interaction with viralproteins, for example Gag proteins. The ligase then ubiquitinates viraland/or cellular proteins that are part of the membrane remodelingmachinery. For example, a number of Gag protein motifs such as PxxP,PxxY, PPXY and YxxL, are known to recruit proteins involved in viralmaturation.

To this end, in certain embodiments, the invention provides a family ofnucleic acid sequences and proteins encoded thereby that play a role inviral maturation: the Alternate Viral Maturation Scaffolding Protein, orthe AVMSP family of proteins. Broadly, AVMSP polypeptides comprise afirst domain or functional role and a second domain. The first domain orfunctional role is selected from the following: SH2, SH3, or membranespanning (“membrane”), or functions as a receptor. A preferred AVMSPalso comprises a second domain that is a RING domain. Accordingly,different categories of AVMSPs may be referred to as RING-SH3 proteinsand nucleic acids, RING-SH2 proteins and nucleic acids, RING-membraneproteins and nucleic acids and RING-receptor polypeptides and nucleicacids. The first domain and second domain may be found in any orderwithin the AVMSP sequence (i.e. the first domain need not be N-terminalto the second domain). It is understood that polypeptides that functionas receptors will often have membrane or other domains. In certainembodiments AVMSP proteins comprise a C2 domain.

In further aspects, in cells infected with viruses that utilize aGag-dependent pathway for assembly, budding and/or release, AVMSPs, actto assemble complexes of proteins that mediate release. AVMSP complexesmay, for example, stimulate, ubiquitylation of certain proteins,stimulate membrane fusion, stimulate assembly of viral particles, or acombination of the preceding. As one of skill in the art can readilyappreciate, any single AVMSP may form multiple different complexes atdifferent times.

In additional aspects, the invention provides nucleic acid sequences andproteins encoded thereby, as well as probes derived from the nucleicacid sequences, antibodies directed to the encoded proteins, diagnosticmethods for detecting cells infected with a virus, and assays foridentifying agents having an antiviral activity.

In one aspect, the invention provides a RING-SH3 nucleic acid, such asan isolated nucleic acid comprising a nucleotide sequence whichhybridizes under stringent conditions to a sequence encoding a RING-SH3protein, such as a sequence of SEQ ID Nos: 1-3, or a sequencecomplementary thereto. In a related embodiment, the nucleic acid is atleast about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequencecorresponding to at least about 12, at least about 15, at least about25, or at least about 40 consecutive nucleotides up to the full lengthof one of SEQ ID Nos. 40-99 or a sequence complementary thereto or up tothe full length of the gene of which said sequence is a fragment. In afurther embodiment, the RING-SH3 nucleic acid comprises a nucleic acidencoding an amino acid sequence as set forth in SEQ ID Nos. 1-39 or anucleic acid complement thereof. In a related embodiment, the encodedamino acid sequence is at least about 80%, 90%, 95%, or 97-98%, or 100%identical to a sequence corresponding to at least about 12, at leastabout 15, at least about 25, or at least about 40 consecutive aminoacids up to the full length of one of SEQ ID Nos: 1-39. In yet anotherembodiment, the RING-SH3 nucleic acid is an isolated nucleic acidencoding a polypeptide comprising a RING domain and an SH3 domain. In apreferred embodiment, the RING-SH3 nucleic acid is a PRT3 nucleic acidof SEQ ID NOs:40-44 or a functional variant thereof.

In a further aspect, the invention provides a RING-SH2 nucleic acid,such as an isolated nucleic acid comprising a nucleotide sequence whichhybridizes under stringent conditions to a sequence encoding a RING-SH2protein, such as a sequence of SEQ ID Nos: 45-46, or a sequencecomplementary thereto. In a related embodiment, the nucleic acid is atleast about 80%, 90%, 95%, or 97-98%, or 100% identical to a sequencecorresponding to at least about 12, at least about 15, at least about25, or at least about 40 consecutive nucleotides up to the full lengthof one of SEQ ID Nos.45-46, or a sequence complementary thereto or up tothe full length of the gene of which said sequence is a fragment. In afurther embodiment, the RING-SH2 nucleic acid comprises a nucleic acidencoding an amino acid sequence as set forth in SEQ ID Nos. 4-5, or anucleic acid complement thereof. In a related embodiment, the encodedamino acid sequence is at least about 80%, 90%, 95%, or 97-98%, or 100%identical to a sequence corresponding to at least about 12, at leastabout 15, at least about 25, or at least about 40 consecutive aminoacids up to the full length of one of SEQ ID NOS: 4-5. In yet anotherembodiment, the RING-SH2 nucleic acid is an isolated nucleic acidencoding a polypeptide comprising a RING domain and an SH2 domain.

In a further aspect, the invention provides a RING-membrane nucleicacid, such as an isolated nucleic acid comprising a nucleotide sequencewhich hybridizes under stringent conditions to a sequence encoding aRING-membrane protein, such as a sequence of SEQ ID Nos: 47-56, or asequence complementary thereto. In a related embodiment, the nucleicacid is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to asequence corresponding to at least about 12, at least about 15, at leastabout 25, or at least about 40 consecutive nucleotides up to the fulllength of one of SEQ ID Nos. 47-56, or a sequence complementary theretoor up to the fill length of the gene of which said sequence is afragment. In a further embodiment, the RING-membrane nucleic acidcomprises a nucleic acid encoding an amino acid sequence as set forth inSEQ ID Nos. 6-15, or a nucleic acid complement thereof. In a relatedembodiment, the encoded amino acid sequence is at least about 80%, 90%,95%, or 97-98%, or 100% identical to a sequence corresponding to atleast about 12, at least about 15, at least about 25, or at least about40 consecutive amino acids up to the full length of one of SEQ ID NOS:6-15. In yet another embodiment, the RING-membrane nucleic acid is anisolated nucleic acid encoding a polypeptide comprising a RING domainand a membrane domain.

In a further aspect, the invention provides a RING-receptor nucleicacid, such as an isolated nucleic acid comprising a nucleotide sequencewhich hybridizes under stringent conditions to a sequence encoding aRING-receptor protein, such as a sequence of SEQ ID Nos: 57-72, or asequence complementary thereto. In a related embodiment, the nucleicacid is at least about 80%, 90%, 95%, or 97-98%, or 100% identical to asequence corresponding to at least about 12, at least about 15, at leastabout 25, or at least about 40 consecutive nucleotides up to the fulllength of one of SEQ ID Nos. 57-72, or a sequence complementary theretoor up to the full length of the gene of which said sequence is afragment. In a further embodiment, the RING-receptor nucleic acidcomprises a nucleic acid encoding an amino acid sequence as set forth inSEQ ID Nos. 16-31, or a nucleic acid complement thereof. In a relatedembodiment, the encoded amino acid sequence is at least about 80%, 90%,95%, or 97-98%, or 100% identical to a sequence corresponding to atleast about 12, at least about 15, at least about 25, or at least about40 consecutive amino acids up to the full length of one of SEQ ID NOS:16-31. In yet another embodiment, the RING-receptor nucleic acid is anisolated nucleic acid encoding a polypeptide comprising a RING domainand a receptor domain.

In one embodiment, the invention provides an expressible RING-SH3,RING-SH2, RING-membrane or RING-receptor nucleic acid operably linked toa transcriptional regulatory sequence, rendering the expressiblenucleotide sequence suitable for use as an expression vector. In anotherembodiment, the nucleic acid may be included in an expression vectorcapable of replicating in a prokaryotic or eukaryotic cell. In a relatedembodiment, the invention provides a host cell transfected with theexpression vector.

In yet another embodiment, the invention provides a substantially pureRING-SH3, RING-SH2, RING-membrane or RING-receptor nucleic acid whichhybridizes under stringent conditions to a nucleic acid probecorresponding to at least about 12, at least about 15, at least about25, or at least about 40 consecutive nucleotides up to the full lengthof one of SEQ ID Nos. 40-72, or a sequence complementary thereto or upto the full length of the gene of which said sequence is a fragment. Theinvention also provides an antisense oligonucleotide analog whichhybridizes under stringent conditions to at least 12, at least 25, or atleast 50 consecutive nucleotides of one of SEQ ID NOS 40-72, or asequence complementary thereto.

In another embodiment, the invention provides a probe/primer comprisinga substantially purified RING-SH3 oligonucleotide, said oligonucleotidecontaining a region of nucleotide sequence which hybridizes understringent conditions to at least about 12, at least about 15, at leastabout 25, or at least about 40 consecutive nucleotides of sense orantisense sequence selected from SEQ ID Nos.40-44, or a sequencecomplementary thereto.

In another embodiment, the invention provides a probe/primer comprisinga substantially purified RING-SH2 oligonucleotide, said oligonucleotidecontaining a region of nucleotide sequence which hybridizes understringent conditions to at least about 12, at least about 15, at leastabout 25, or at least about 40 consecutive nucleotides of sense orantisense sequence selected from SEQ ID Nos.4546, or a sequencecomplementary thereto.

In another embodiment, the invention provides a probe/primer comprisinga substantially purified RING-membrane oligonucleotide, saidoligonucleotide containing a region of nucleotide sequence whichhybridizes under stringent conditions to at least about 12, at leastabout 15, at least about 25, or at least about 40 consecutivenucleotides of sense or antisense sequence selected from SEQ DNos.47-56, or a sequence complementary thereto.

In another embodiment, the invention provides a probe/primer comprisinga substantially purified RING-receptor oligonucleotide, saidoligonucleotide containing a region of nucleotide sequence whichhybridizes under stringent conditions to at least about 12, at leastabout 15, at least about 25, or at least about 40 consecutivenucleotides of sense or antisense sequence selected from SEQ IDNos.57-72, or a sequence complementary thereto.

In preferred embodiments, a probe as described above selectivelyhybridizes with a target nucleic acid. In another embodiment, the probemay include a label group attached thereto and able to be detected. Thelabel group may be selected from radioisotopes, fluorescent compounds,enzymes, and enzyme co-factors. The invention further provides arrays ofat least about 10, at least about 25, at least about 50, or at leastabout 100 different probes as described above attached to a solidsupport.

In another aspect, the invention provides polypeptides. In oneembodiment, the invention pertains to a RING-SH3 polypeptide includingan amino acid sequence encoded by a nucleic acid comprising a nucleotidesequence which hybridizes under stringent conditions to a sequence ofSEQ ID Nos. 40-44, or a sequence complementary thereto, or a fragmentcomprising at least about 25, or at least about 40 amino acids thereof.

In another aspect, the invention provides polypeptides. In oneembodiment, the invention pertains to a RING-SH2 polypeptide includingan amino acid sequence encoded by a nucleic acid comprising a nucleotidesequence which hybridizes under stringent conditions to a sequence ofSEQ ID Nos. 45-46, or a sequence complementary thereto, or a fragmentcomprising at least about 25, or at least about 40 amino acids thereof.

In another aspect, the invention provides polypeptides. In oneembodiment, the invention pertains to a RING-membrane polypeptideincluding an amino acid sequence encoded by a nucleic acid comprising anucleotide sequence which hybridizes under stringent conditions to asequence of SEQ ID Nos. 47-56, or a sequence complementary thereto, or afragment comprising at least about 25, or at least about 40 amino acidsthereof.

In another aspect, the invention provides polypeptides. In oneembodiment, the invention pertains to a RING-receptor polypeptideincluding an amino acid sequence encoded by a nucleic acid comprising anucleotide sequence which hybridizes under stringent conditions to asequence of SEQ ID Nos. 57-72, or a sequence complementary thereto, or afragment comprising at least about 25, or at least about 40 amino acidsthereof.

In a preferred embodiment, the polypeptide is identical with orhomologous to a RING-SH3, RING-SH2, RING-membrane or RING-receptorprotein represented by SEQ ID Nos: 1-31. For instance, a polypeptidepreferably has an amino acid sequence at least 70% homologous to apolypeptide represented by any of SEQ ID Nos: 1-31, though polypeptideswith higher sequence homologies of, for example, 80%, 90% or 95% arealso contemplated. The polypeptide can comprise a full length protein,such as represented in the sequence listings, or it can comprise afragment of, for instance, at least 5, 10, 20, 50, 100, 150 or 200 aminoacids in length.

In another preferred embodiment, the invention features a purified orrecombinant polypeptide fragment of a RING-SH3, RING-SH2, RING-membraneor RING-receptor polypeptide, which polypeptide has the ability tomodulate, e.g., mimic or antagonize, an activity of a wild-typeRING-SH3, RING-SH2, RING-membrane or RING-receptor polypeptide.Preferably, the polypeptide fragment comprises a sequence identical orhomologous to an amino acid sequence designated in one of SEQ ID Nos:1-31.

Moreover, as described below, the RING-SH3, RING-SH2, RING-membrane orRING-receptor polypeptide can be either an agonist (e.g. mimics), oralternatively, an antagonist of a biological activity of a naturallyoccurring form of the protein, e.g., the polypeptide is able to modulatethe intrinsic biological activity of a RING-SH3, RING-SH2, RING-membraneor RING-receptor complex, such as an enzymatic activity, binding toother cellular components, cellular compartmentalization, and the like.

The subject proteins can also be provided as chimeric molecules, such asin the form of fusion proteins. For instance, the AVMSP can be providedas a recombinant fusion protein which includes a second polypeptideportion, e.g., a second polypeptide having an amino acid sequenceunrelated (heterologous) to the AVMSP, e.g. the second polypeptideportion is glutathione-S-transferase, e.g. the second polypeptideportion is an enzymatic activity such as alkaline phosphatase, e.g. thesecond polypeptide portion is an epitope tag, e.g. the secondpolypeptide is an affinity purification tag.

Yet another aspect of the present invention concerns an immunogencomprising an AVMSP in an immunogenic preparation, the immunogen beingcapable of eliciting an immune response specific for said AVMSP; e.g. ahumoral response, e.g. an antibody response; e.g. a cellular response.In preferred embodiments, the immunogen comprising an antigenicdeterminant, e.g. a unique determinant, from a protein represented byone of SEQ ID Nos. 1-39.

In yet another aspect, this invention provides antibodies immunoreactivewith one or more AVMSPs. In one embodiment, antibodies are specific fora RING domain, an SH3 domain, a SH2 domain, or a receptor domain andpreferably the domain is part of an AVMSP. In a more specificembodiment, the domain is part of an amino acid sequence set forth inSEQ ID Nos. 1-39. In another embodiment, the antibodies areimmunoreactive with one or more proteins having an amino acid sequencethat is at least 80% identical to an amino acid sequence as set forth inSEQ ID Nos. 1-39. In other embodiments, an antibody is immunoreactivewith one or more proteins having an amino acid sequence that is 85%,90%, 95%, 98%, 99% or identical to an amino acid sequence as set forthin SEQ ID Nos. 1-39.

In an additional aspect, the invention provides complexes comprising anAVMSP and an AVMSP associated protein (an “AVMSP-AP”). In oneembodiment, the invention provides an isolated protein complexcomprising a RING-SH3, RING-SH2, RING-membrane or RING-receptorpolypeptide in combination with at least one polypeptide selected fromthe group consisting of: a RING-SH3, a RING-SH2, a RING-membrane, aRING-receptor, a Gag protein, a Gag late domain, PI3K, actin, myosin,Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2enzyme, tsg101, a cullin and a clathrin. In another embodiment, theisolated protein complex comprises a RING-SH3, RING-SH2, RING-membraneor RING-receptor polypeptide and a Gag protein in combination with apolypeptide selected from the group consisting of: a RING-SH3, aRING-SH2, a RING-membrane, a RING-receptor, PI3K, actin, myosin, Hsp60,Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme,tsg101, a cullin and a clathrin.

In yet another embodiment, the invention provides an isolated proteincomplex comprising an AVMSP polypeptide and a HIV Gag protein incombination with a polypeptide selected from the group consisting of:RING-SH3, RING-SH2, RING-membrane, RING-receptor, PI3K, actin, myosin,Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2enzyme, tsg101, a cullin and a clathrin. The invention also provides anisolated protein complex comprising a RING-SH3, RING-SH2, RING-membraneor RING-receptor polypeptide and an HIV Gag protein in combination witha polypeptide selected from the group consisting of: RING-SH3, RING-SH2,RING-membrane or RING-receptor, PI3K, actin, myosin, Hsp60, Hsp70,Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, acullin and a clathrin.

In yet another aspect, the invention provides an assay for screeningtest compounds for inhibitors, or alternatively, potentiators, of aninteraction between an AVMSP and an AVMSP-AP. In the case of a RING-SH3polypeptide, exemplary associated proteins (“RING-SH3-AP”) includeRING-SH3 proteins, E2 proteins (e.g. tsg101), Gag proteins, proteinscomprising an L-domain, phosphatidylinositol-3-kinases, as well asproteins involved in endocytosis such as clathrins, actins, myosins,HSP60, HSP70, HSP90, STAM1, STAM2A, and STAM2B. An exemplary methodincludes the steps of (i) combining AVMSP-AP (e.g. a RING-SH3-AP,RING-SH2-AP, RING-membrane-AP or RING-receptor-AP), an AVMSP, and a testcompound, e.g., under conditions (including, as desired, the addition ofadditional proteins) wherein, but for the test compound, the AVMSP andan AVMSP-AP are able to interact; and (ii) detecting the formation of acomplex which includes the AVMSP and an AVMSP-AP. A statisticallysignificant change, such as a decrease, in the formation of the complexin the presence of a test compound (relative to what is seen in theabsence of the test compound) is indicative of a modulation, e.g.,inhibition, of the interaction between the AVMSP and an AVMSP-AP.Similar assays may employ preformed AVMSP-AVMSP-AP complexes to assessthe ability of the test compound to destabilize or stabilize thecomplex.

In yet another aspect, the invention provides cells carrying arecombinant form of an AVMSP nucleic acid, often included on a vector.In further embodiments, cells carry a recombinant form of an AVMSPnucleic acid and a recombinant form of a nucleic acid encoding a Gagprotein and/or a polypeptide comprising an L domain motif, such asP(T/S)AP, PPxY or YxxL. In certain aspects, the cells are bacterial, andin other aspects the cells are eukaryotic cells, preferrably a mammaliancell line.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art.

Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Human PRT3 amino acid sequence (SEQ ID NO: 1)

FIG. 2A: N terminus protein fragment of hPRT3 (public gi: 10432612; SEQID NO:2)

FIG. 2B: C terminus protein fragment of hPRT3 (public gi: 7959249; SEQID NO:39)

FIGS. 3-39 show amino acid sequences for examples of AVMSP polypeptides.

FIGS. 40-78 show nucleic acid sequences encoding examples of AVMSPpolypeptides.

FIG. 79: Human PRT3 full mRNA, annotated sequence

FIG. 80: Domain analysis of human PRT3

FIG. 81: Diagram of human PRT3 nucleic acids. The diagram shows thefull-length PRT3 gene and the position of regions amplified by RT-PCR ortargeted by siRNA used in FIG. 11.

FIG. 82: Knockdown of PRT3 mRNA by siRNA duplexes. HeLa SS-6 cells weretransfected with siRNA against Lamin A/C (lanes 1, 2) or PRT3 (lanes3-10). PRT3 siRNA was directed against the coding region (153—lanes 3,4;155—lanes 5,6) or the 3′UTR (157-lanes 7, 8; 159—lanes 9, 10). Cellswere harvested 24 hours post-transfection, RNA extracted, and PRT3 mRNAlevels compared by RT-PCR of a discrete sequence in the coding region ofthe PRT3 gene (see FIG. 10). GAPDH is used an RT-PCR control in eachreaction.

FIG. 83: PRT3 affects the release of VLP from cells. A) Posphohimages ofSDS-PAGE gels of immunoprecipitations of ³⁵S pulse-chase labeled Gagproteins are presented for cell and viral lysates from transfected HeLacells that where either untreated or treated with PRT3 RNAi (50 nM for48 hours). The time during the chase period (1,2,3,4 and 5 after thepulse) are presented from left to right for each image.

FIG. 84: Release of VLP from cells at steady state. Hela cells weretransfected with an “HV-encoding plasmid and siRNA. Lanes 1, 3 and 4were transfected with wild-type HIV-encoding plasmid. Lane 2 wastransfected with an HI-V-encoding plasmids which contains a pointmutation in p6 (PTAP to ATAP). Control siRNA (lamin A/C) was transfectedto cells in lanes 1 and 2. siRNA to Tsg101 was transfected in lane 4 andsiRNA to PRT3 in lane 3.

FIG. 85: Exemplary HIV-1 Gag Nucleic Acid Sequence (Acc. No. NC-001802)[SEQ ID NO:80]

FIG. 86: Exemplary HIV-1 Gag Amino Acid Sequence (Acc. No. NP_(—)057850)[SEQ ID NO:81]

FIG. 87: Exemplary HIV-I p6 Amino Acid Sequence (SEQ ID NO:82)

FIG. 88: Representative consensus terms for domains

FIG. 89: Comparative Sequence Analysis—Amino Acid Grouping

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

The term “Alternate Viral Maturation Scaffolding Protein” or “AVMSP” isused herein to indicate a polypeptide comprising a first domain orfunctionality and a second domain. The first domain or functionality isselected from the group consisting of: an SH2 domain, an SH3 domain, amembrane spanning domain, and a receptor functionality (meaning theprotein, or a portion thereof functions as a receptor). An AVMSP alsocomprises a second domain that is, for the purposes of this application,a RING domain. The first domain and second domain may be found in anyorder within the AVMSP sequence (i.e. the first domain need not beN-terminal to the second domain). The term AVMSP encompasses proteinsrepresented as SEQ ID Nos. 1-39. It is understood that polypeptideshaving functional roles, such as receptors, may farther comprisemembrane or other domains. In other embodiments, AVMSPs may furthercomprise one or more C2 domains. In cells infected with viruses thatutilize a Gag-dependent pathway for budding and release, an AVMSP mayact to assemble complexes of proteins that mediate release. AVMSPcomplexes may stimulate ubiquitylation of certain proteins or stimulatemembrane fusion or both. Any single AVMSP may form multiple differentAVMSP complexes at different times.

The term “Alternate Viral Maturation Scaffolding Protein-AssociatedProtein” (AVMSP-AP) refers to protein capable of interacting and/orbinding to an AVMSP. Generally, the AVMSP-AP may interact directly orindirectly with the AVMSP. Examples of these proteins include forexample the “Late domain” or “L domain”, which is a small portion of aGag protein that promotes efficient release of virion particles from themembrane of the host cell. L domains typically comprise one or moreshort motifs (L motifs). Exemplary sequences include: P(T/S)AP, PxxL,PPxY (eg. PPPY), YxxL (eg. YPDL), PxxP. Other exemplary AVMSP-APs areprovided throughout.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, ionic and/orhydrogen-bond interactions under physiological conditions.

A “C2 domain” is a calcium binding domain. Certain C2 domains comprisethe consensus sequence set forth in FIG. 14 as “Consensus/80%”. Other C2domains comprise the consensus sequences set forth as “Consensus/65%” or“Consensus/50%”.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding a polypeptide with a second amino acid sequencedefining a domain foreign to and not substantially homologous with anydomain of the first amino acid sequence. A chimeric protein may presenta foreign domain which is found (albeit in a different protein) in anorganism which also expresses the first protein, or it may be an“interspecies”, “intergenic”, etc. fusion of protein structuresexpressed by different kinds of organisms.

The terms “compound”, “test compound” and “molecule” are used hereininterchangeably and are meant to include, but are not limited to,peptides, nucleic acids, carbohydrates, small organic molecules, naturalproduct extract libraries, and any other molecules (including, but notlimited to, chemicals, metals and organometallic compounds).

The phrase “conservative amino acid substitution” refers to grouping ofamino acids on the basis of certain common properties. A functional wayto define common properties between individual amino acids is to analyzethe normalized frequencies of amino acid changes between correspondingproteins of homologous organisms (Schulz, G. E. and R. H. Schirmer.,Principles of Protein Structure, Springer-Verlag). According to suchanalyses, groups of amino acids may be defined where amino acids withina group exchange preferentially with each other, and therefore resembleeach other most in their impact on the overall protein structure(Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure,Springer-Verlag). Examples of amino acid groups defined in this mannerinclude:

-   (i) a charged group, consisting of Glu and Asp, Lys, Arg and His,-   (ii) a positively-charged group, consisting of Lys, Arg and His,-   (iii) a negatively-charged group, consisting of Glu and Asp,-   (iv) an aromatic group, consisting of Phe, Tyr and Trp,-   (v) a nitrogen ring group, consisting of His and Trp,-   (vi) a large aliphatic nonpolar group, consisting of Val, Leu and    Ile,-   (vii) a slightly-polar group, consisting of Met and Cys,-   (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly,    Ala, Glu, Gln and Pro,-   (ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys,    and-   (x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups presented above, each amino acid residue mayform its own group, and the group formed by an individual amino acid maybe referred to simply by the one and/or three letter abbreviation forthat amino acid commonly used in the art.

A “conserved residue” is an amino acid that is relatively invariantacross a range of similar proteins. Often conserved residues will varyonly by being replaced with a similar amino acid, as described above for“conservative amino acid substitution”.

The term “domain” as used herein refers to a region of a protein thatcomprises a particular structure and/or performs a particular function.

The term “Gag protein” or “Gag polypeptide” refers to a polypeptidehaving Gag activity and preferably comprising an L (or late) domain.Exemplary Gag proteins include a motif such as PXXP, PPXY, PXXY, YXXL,RXXPXXP, RPDPTAP, RPLPVAP, RPEPTAP, PTAPPEY, PTAPPEE and/or RPEPTAPPEE.An exemplary HIV-1 Gag protein is SEQ ID NO: 32. Typically, an HIV Gagprotein comprises a p6 protein.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated” or“non-homologous” shares less than 40% identity, though preferably lessthan 25% identity with a sequence of the present invention. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention may be used as a “query sequence” to perform asearch against public databases to, for example, identify other familymembers, related sequences or homologs. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.(1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score—100, wordlength=12 to obtainnucleotide sequences homologous to nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used. See http://www.ncbi.nlm.nih.gov.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Watermanalgorithm may also be used to determine identity.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “intron” refers to a portion of nucleic acid that is intiallytranscribed into RNA but later removed such that it is not, for the mostpart, represented in the processed mRNA. Intron removal occurs throughreactions at the 5′ and 3′ ends, typically referred to as 5′ and 3′splice sites, respectively. Alternate use of different splice sitesresults in splice variants. An intron is not necessarily situatedbetween two “exons”, or portions that code for amino acids, but mayinstead be positioned, for example, between the promoter and the firstexon. An intron may be self-splicing or may require cellular componentsto be spliced out of the mRNA. A “heterologous intron” is an intron thatis inserted into a coding sequence that is not naturally associated withthat coding sequence. In addition, a heterologous intron may be agenerally natural intron wherein one or both of the splice sites havebeen altered to provide a desired quality, such as increased ordescreased splice efficiency. Heterologous introns are often inserted,for example, to improve expression of a gene in a heterologous host, orto increase the production of one splice variant relative to another. Asan example, the rabbit beta-globin gene may be used, and is commerciallyavailable on the pCI vector from Promega Inc. Other exemplary intronsare provided in Lacy-Hulbert et al. (2001) Gene Ther 8(8):649-53.

The term “isolated”, as used herein with reference to the subjectproteins and protein complexes, refers to a preparation of protein orprotein complex that is essentially free from contaminating proteinsthat normally would be present with the protein or complex, e.g., in thecellular milieu in which the protein or complex is found endogenously.Thus, an isolated protein complex is isolated from cellular componentsthat normally would “contaminate” or interfere with the study of thecomplex in isolation, for instance while screening for modulatorsthereof. It is to be understood, however, that such an “isolated”complex may incorporate other proteins the modulation of which, by thesubject protein or protein complex, is being investigated.

The term “isolated” as also used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules in a form which does not occurin nature. Moreover, an “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotides.

The term “maturation” as used herein refers to the processing of viralproteins leading to the pinching off of nascent virion from the cellmembrane, including, for example, assembly, budding and release.

A “membrane associated protein” is meant to include proteins that areintegral membrane proteins as well as proteins that are stablyassociated with a membrane.

A “membrane spanning domain” or “transmembrane domain” or membranedomain” is a domain that traverses a biological lipid bilayer membranefrom one side to the other. Generally membrane domains are identified asa region of a protein having a high hydrophobicity. Membrane domains aretypically between 15 and 25 amino acids in length. Exemplary methods foridentifying membrane domains are provided in FIG. 129.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

The term “p6” or p6gag” is used herein to refer to an HIV proteincomprising a viral L domain. Antibodies that bind to a p6 domain arereferred to as “anti-p6 antibodies”. p6 also refers to proteins thatcomprise artificially engineered L domains including, for example, Ldomains comprising a series of L motifs. An exemplary HIV-1 p6 is SEQ IDNO: 85.

A “profile” is used herein to indicate an aggregate of informationregarding a preparation of cell or membrane surface proteins. A profilewill comprise, at minimum, information regarding the presence or absenceof such proteins. More typically, a profile will comprise informationregarding the presence or absence of a plurality of such proteins. Inaddition, a profile may contain other information about each identifiedprotein, such as relative or absolute amount of protein present, thedegree of post-translational modification, membrane topology,three-dimensional structure, isoelectric point, molecular weight, etc. A“test profile” is a profile obtained from a subject of unknowndiagnostic state. A “reference profile” is a profile obtained fromsubject known to be infected or uninfected.

The terms peptides, proteins and polypeptides are used interchangeablyherein.

The term “purified protein” refers to a preparation of a protein orproteins which are preferably isolated from, or otherwise substantiallyfree of, other proteins normally associated with the protein(s) in acell or cell lysate. The term “substantially free of other cellularproteins” (also referred to herein as “substantially free of othercontaminating proteins”) is defined as encompassing individualpreparations of each of the component proteins comprising less than 20%(by dry weight) contaminating protein, and preferably comprises lessthan 5% contaminating protein. Functional forms of each of the componentproteins can be prepared as purified preparations by using a cloned geneas described in the attached examples. By “purified”, it is meant, whenreferring to component protein preparations used to generate areconstituted protein mixture, that the indicated molecule is present inthe substantial absence of other biological macromolecules, such asother proteins (particularly other proteins which may substantiallymask, diminish, confuse or alter the characteristics of the componentproteins either as purified preparations or in their function in thesubject reconstituted mixture). The term “purified” as used hereinpreferably means at least 80% by dry weight, more preferably in therange of 85% by weight, more preferably 95-99% by weight, and mostpreferably at least 99.8% by weight, of biological macromolecules of thesame type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “pure” as used herein preferably has the samenumerical limits as “purified” immediately above.

A “receptor” or “protein having a receptor function” is a protein thatinteracts with an extracellular ligand or a ligand that is within thecell but in a space that is topologically equivalent to theextracellular space (eg. inside the Golgi, inside the endoplasmicreticulum, inside the nuclear membrane, inside a lysosome or transportvesicle, etc.). Exemplary receptors are identified herein by annotationas such in various public databases. Receptors often have membranedomains.

A “recombinant nucleic acid” is any nucleic acid that has been placedadjacent to another nucleic acid by recombinant DNA techniques. A“recombined nucleic acid” also includes any nucleic acid that has beenplaced next to a second nucleic acid by a laboratory genetic techniquesuch as, for example, tranformation and integration, transposon hoppingor viral insertion. In general, a recombined nucleic acid is notnaturally located adjacent to the second nucleic acid.

The term “recombinant protein” refers to a protein of the presentinvention which is produced by recombinant DNA techniques, whereingenerally DNA encoding the expressed protein is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the heterologous protein. Moreover, the phrase “derived from”,with respect to a recombinant gene encoding the recombinant protein ismeant to include within the meaning of “recombinant protein” thoseproteins having an amino acid sequence of a native protein, or an aminoacid sequence similar thereto which is generated by mutations includingsubstitutions and deletions of a naturally occurring protein.

A “RING domain” or “Ring Finger” is a zinc-binding domain with a definedoctet of cysteine and histidine residues. Certain RING domains comprisethe consensus sequences as set forth below (amino acid nomenclature isas set forth in Table 1): Cys Xaa Xaa Cys Xaa₁₀₋₂₀ Cys Xaa His Xaa₂₋₅Cys Xaa Xaa Cys Xaa₁₃₋₅₀ Cys Xaa Xaa Cys or Cys Xaa Xaa Cys Xaa₁₀₋₂₀ CysXaa His Xaa₂₋₅ His Xaa Xaa Cys Xaa₁₃₋₅₀ Cys Xaa Xaa Cys. Preferred RINGdomains of the invention bind to various protein partners to form acomplex that has ubiquitin ligase activity. RING domains preferablyinteract with at least one of the following protein types: F boxproteins, E2 ubiquitin conjugating enzymes and cullins.

A “scaffolding protein” is a protein that brings together two or moredifferent proteins that interact to accomplish one or more particularfunctions. A scaffolding protein may, in addition to acting as ascaffold, carry out biochemical functions on its own or as part of acomplex.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 2.5 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing or inorganic molecules. Many pharmaceutical companieshave extensive libraries of chemical and/or biological mixturescomprising arrays of small molecules, often fungal, bacterial, or algalextracts, which can be screened with any of the assays of the invention.

An “SH2” or “Src Homology 2” domain is a protein domain of generallyabout 100 amino acid residues. SH2 domains function as regulatorymodules of intracellular signalling cascades by interacting with highaffinity to phosphotyrosine-containing target peptides in asequence-specific and phosphorylation-dependent manner.

An “SH3” or “Src Homology 3” domain is a protein domain of generallyabout 60 amino acid residues first identified as a conserved sequence inthe non-catalytic part of several cytoplasmic protein tyrosine kinases(e.g. Src, Abl, Lck). SH3 domains mediate assembly of specific proteincomplexes via binding to proline-rich peptides.

As used herein, the term “specifically hybridizes” refers to the abilityof a nucleic acid probe/primer of the invention to hybridize to at least12, 15, 20, 25, 30, 35, 40, 45, 50 or 100 consecutive nucleotides of atarget gene sequence, or a sequence complementary thereto, or naturallyoccurring mutants thereof, such that it has less than 15%, preferablyless than 10%, and more preferably less than 5% background hybridizationto a cellular nucleic acid (e.g., mRNA or genomic DNA) other than thetarget gene. A variety of hybridization conditions may be used to detectspecific hybridization, and the stringency is determined primarily bythe wash stage of the hybridization assay. Generally high temperaturesand low salt concentrations give high stringency, while low temperaturesand high salt concentrations give low stringency. Low stringencyhybridization is achieved by washing in, for example, about 2.0×SSC at50° C., and high stringency is acheived with about 0.2×SSC at 50° C.Further descriptions of stringency are provided below.

“STAM” proteins include a family of proteins involved in receptormediated exo- and endocytosis as well as cellular signalling, generally.STAM proteins generally comprise an N-terminal VHS homology domain, aubiquitin-interacting motif and an SH3 domain and optionally animmunoreceptor tyrosine-based activation motif. STAM 1 and STAM 2A areinvolved in cytokine-mediated signalling for DNA synthesis and c-mycinduction. EAST and STAM 2A/Hbp play a role in receptor-mediated endo-and exocytosis and probably also in the regulation of actincytoskeleton. (Lohi et al. FEBS Lett 2001 Nov. 23;508(3):287-90).

As applied to polypeptides, “substantial sequence identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap which share at least 90 percentsequence identity, preferably at least 95 percent sequence identity,more preferably at least 99 percent sequence identity or more.Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For example, the substitution ofamino acids having similar chemical properties such as charge orpolarity are not likely to effect the properties of a protein. Examplesinclude glutamine for asparagine or glutamic acid for aspartic acid.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of a recombinant protein gene isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring form of the protein.

A “UIM” domain is a ubiquitin binding motif.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome. In the present specification, “plasmid”and “vector” are used interchangeably as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors which serve equivalentfunctions and which become known in the art subsequently hereto.

A “virion” is a complete viral particle; nucleic acid and capsid (and alipid envelope in some viruses.

A “VHS” domain is a “Vps27p, Hrs and STAM” domain, named for theproteins in which it has been identified, and includes a DXXLL sequencemotif. VHS domains have also been identified in the GGA(Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding)proteins. In certain embodiments, VHS domains of the invention recognizeone or more acidic-cluster-dileucine signals found in the cytoplasmictails of sorting receptors, such as mannose-6-phosphate receptors.(Misra et al. (2002) Nature 2002 Feb. 21;415(6874):933-7) TABLE 1Abbreviations for classes of amino acids* Symbol Category Amino AcidsRepresented X1 Alcohol Ser, Thr X2 Aliphatic Ile, Leu, Val Xaa Any Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, Tyr X4 Aromatic Phe, His, Trp, Tyr X5 Charged Asp,Glu, His, Lys, Arg X6 Hydrophobic Ala, Cys, Phe, Gly, His, Ile, Lys,Leu, Met, Thr, Val, Trp, Tyr X7 Negative Asp, Glu X8 Polar Cys, Asp,Glu, His, Lys, Asn, Gln, Arg, Ser, Thr X9 Positive His, Lys, Arg X10Small Ala, Cys, Asp, Gly, Asn, Pro, Ser, Thr, Val X11 Tiny Ala, Gly, SerX12 Turnlike Ala, Cys, Asp, Glu, Gly, His, Lys, Asn, Gln, Arg, Ser, ThrX13 Asparagine-Aspartate Asn, Asp*Abbreviations as adopted fromhttp://smart.embl-heidelberg.de/SMART_DATA/alignments/consensus/grouping.html.2. Overview

In certain aspects, the invention relates to the observation that AVMSPpolypeptides are involved in viral maturation process such as assembly,budding and/or release. Any one AVMSP may be involved at one or morestages of viral maturation and may form one or more complexes with viraland/or host proteins.

In certain aspects, AVMSP sequences disclosed herein, and relatedmethods and compositions may be used to manipulate a variety ofsignificant biological processes. In certain embodiments, AVMSP nucleicacids and polypeptides of the invention are involved in a virallifecycle. In one embodiment, an SH3 domain of a RING-SH3 protein bindsto a viral Gag sequence represented by a consensus sequence P(T/S)AP,RXXP(T/S)AP or PXXDY, such as, for example, the PTAP or PFRDY sequencesof HIV Gag (positioned, for example, at 455458 and 292-296,respectively).

In a further embodiment, AVMSP polypeptides may be involved in theformation of endocytosis-like or vesicle trafficking-like complexes thatare involved in a stage of viral maturation. Such complexes may includecomplexes similar to those formed in connection with clathrin-mediatedvesicle trafficking or those formed in connection with coatomer-coatedvesicle trafficking.

Viral assembly, budding and release is expected to require a range ofdifferent protein complexes that incorporate host proteins involved indifferent aspects of vesicle trafficking, including vesicle formationproteins such as ARFs, COPs, RABs and clathrins, cytoskeletal proteinssuch as actins and myosins, cytoskeletal regulators such as Rac and Rho,heat shock proteins, STAM proteins and viral proteins, particularlyproteins having a Late domain, such as Gag. Such complexes are expectedto incorporate one or more AVMSPs, and particularly AVMSPs such asRING-SH3 proteins. At each stage of vesicle transport, the exactcomponents of the relevant complexes may shift, but in general, it isunderstood that disrupting the ability of an AVMSP to participate incomplex formation or dissolution will be effective in disrupting viralproduction. In certain embodiments, it is understood that interferingwith AVMSP activity will not decrease the rate of viral production butwill result in the production of defective viral particles havingdecreased ability to infect another host cell.

It is generally understood that AVMSPs, especially AVMSPs having similarfunctional domains, may exhibit substantial functional overlap in bothnative host functions and in the viral lifecycle. Accordingly, theinvention provides methods for inhibiting a virus by interfering withthe activity of more than one AVMSP. A preferred antiviral agent isable, as a single compound or mixture of compounds to interfere withmore than one AVMSP.

3. Exemplary Nucleic Acids and Expression Vectors

In certain aspects the invention provides nucleic acids encoding AtemateViral Maturation Scaffolding Proteins (AVMSPs)., such as for exampleRING-SH3 proteins, RING-SH2 proteins, RING-membrane proteins andRING-receptor proteins. There are four basic classes of AVMSPs: theRING-SH3 class, comprising at least one SH3 domain and at least one RINGdomain, the RING-SH2 class, comprising at least one SH2 domain and atleast one RING domain, the RING-membrane class, comprising at least oneRING domain and at least one transmembrane domain, and the RING-receptorclass, comprising at least one RING domain and having receptorfunctionality. These four classes are not mutually exclusive, as, forexample, a polypeptide may comprise a RING domain, and SH3 domain and anSH2 domain. In preferred embodiments, proteins of any of the fourclasses comprise at least one C2 domain.

Nucleic acids of the invention is further understood to include nucleicacids that encode variants of AVMSPs. Variant nucleotide sequencesinclude sequences that differ by one or more nucleotide substitutions,additions or deletions, such as allelic variants; and will, therefore,include coding sequences that differ from the nucleotide sequence of thecoding sequence designated in Tables 2-4 e.g., due to the degeneracy ofthe genetic code. Variants will also include nucleotide sequences thathybridize under stringent conditions (i.e., equivalent to about 20-27°C. below the melting temperature (T_(m)) of the DNA duplex formed inabout 1M salt) to the nucleotide sequence of a coding sequencedesignated in Tables 2 and 3. Alternatively put, variants will alsoinclude nucleotide sequences that hybridize under moderately stringentconditions, for example at about 2.0×SSC and about 40° C. to thenucleotide sequence of a coding sequence designated in Tables 2-4. Inanother embodiment, equivalent nucleic acid sequences include sequencesthat will hybridize under highly stringent conditions to a nucleotidesequence of a coding sequence designated in Tables 2-4.

One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

In one embodiment, variants will further include nucleic acid sequencesderived from and evolutionarily related to a nucleotide sequence of acoding sequence designated in Tables 2-4. TABLE 2 Exemplary AVMSPsAccession Nos. Nucleotide Protein Selected Domains or Name Nucleotide;Protein SEQ I.D. No. SEQ I.D. No. Features of Interest Human PRT3 40 1RING, SH3 Coding sequence Human PRT3 41 1 cDNA 5′ cDNA fragment of 42 2human PRT3 3′mRNA fragment of 43 39 human PRT3 SIMILAR TO MUS EMBL;AK021429; 44 3 RING, SH3 MUSCULUS BAB13822.1 PLENTY OF SH3S (POSH)SH3-BINDING EMBL; U26710; 45 4 RING, SH2 PROTEIN CBL-B AAB09291.1SH3-BINDING EMBL; AB028645; 46 5 RING, SH2 PROTEIN CBL-C BAA86298.1MULTIPLE EMBL; AF064801; 47 6 RING, Membrane MEMBRANE AAC39930.1SPANNING RECEPTOR TRC8 AUTOCRINE EMBL; AF124145; 48 7 RING, MembraneMOTILITY FACTOR AAD56722.1 RECEPTOR MEMBRANE- EMBL; AF255303; 49 8 RING,Membrane ASSOCIATED AAG00432.1 NUCLEIC ACID BINDING PROTEIN (FRAGMENT)NADH- EMBL; X81900; 50 9 RING, Membrane UBIQUINONE CAA57489.1OXIDOREDUCTASE MWFE SUBUNIT PEROXISOME EMBL; M86852; 51 10 RING,Membrane ASSEMBLY AAC12785.1 FACTOR-1 (PAF-1) PEROXISOME EMBL; AF060502;52 11 RING, Membrane ASSEMBLY AAC18133.1 PROTEIN 10 PEROXISOME EMBL;U91521; 53 12 RING, Membrane ASSEMBLY AAC68812.1 PROTEIN 12ACETYLCHOLINE EMBL; Z33905; 54 13 RING, Membrane RECEPTOR- CAA83954.1ASSOCIATED 43 KDA PROTEIN PEROXISOMAL EMBL; BC000661; 55 14 RING,Membrane MEMBRANE AAH00661.1 PROTEIN 3 HsRmal EMBL; AB056869; 56 15RING, Membrane BAB39359.1 HTRIP EMBL; U77845; 57 16 RING, ReceptorAAB52993.1 CYSTEIN RICH EMBL; X80200; 58 17 RING, Receptor DOMAINCAA56491.1 ASSOCIATED TO RING AND TRAF PROTEIN PML-RAR EMBL; M73779; 5918 RING, Receptor AAA60126.1 TRAF5 EMBL; AB000509; 60 19 RING, ReceptorBAA25262.1 TUMOR NECROSIS EMBL; AF082185; 61 20 RING, Receptor FACTORAAC32376.1 RECEPTOR- ASSOCIATED FACTOR 4A HFB30 EMBL; AB022663; 62 21RING, Receptor BAA78677.1 BIR2 EMBL; L49432; 63 22 RING, ReceptorAAC41943.1 BIR3 EMBL; L49431; 64 23 RING, Receptor AAC41942.1 CBL EMBL;X57110; 65 24 RING, Receptor CAA40393.1 PML EMBL; M79462; 66 25 RING,Receptor AAA60388.1 RAG1 EMBL; M29474; 67 26 RING, Receptor AAA60248.1RAPSYN EMBL; Z33905; 68 27 RING, Receptor CAA83954.1 TIFI-ALPHA EMBL;AF009353; 69 28 RING, Receptor AAB63585.1 TIFI-GAMMA EMBL; AF119043; 7029 RING, Receptor AAD17259.1 TRAF2 EMBL; U12597; 71 30 RING, ReceptorAAA87706.1 TRAF3 EMBL; U21092; 72 31 RING, Receptor AAC50112.1 HIP116EMBL; L34673; 73 32 RING AAA67436.1 STAF50 EMBL; X82200; 74 33 RINGCAA57684.1 HLTF-1 EMBL; Z46606; 75 34 RING CAA86571.1 BIR4 EMBL; U45880;76 35 RING AAC50373.1 ADENOVIRUS 5 EMBL; X86098; 77 36 RING E1A-BINDINGCAA60052.1 PROTEIN HT2A EMBL; U18543; 78 37 RING AAA86474.1 TRAF3 EMBL;U21092; 79 38 RING AAC50112.1

TABLE 3 Exemplary POSH nucleic acids Accession Sequence Name OrganismNumber cDNA FLJ11367 fis, clone Homo sapiens AK021429 HEMBA1000303Plenty of SH3 domains Mus musculus NM_021506 (POSH) mRNA Plenty of SH3s(POSH) Mus musculus AF030131 mRNA Plenty of SH3s (POSH) Drosophilamelanogaster NM_079052 mRNA Plenty of SH3s (POSH) Drosophilamelanogaster AF220364 mRNA

TABLE 4 Exemplary POSH polypeptides Accession Sequence Name OrganismNumber SH3 domains- Mus musculus T09071 containing protein POSH plentyof SH3 domains Mus musculus NP_067481 Plenty of SH3s; POSH Mus musculusAAC40070 Plenty of SH3s Drosophila melanogaster AAF37265 LD45365pDrosophila melanogaster AAK93408 POSH gene product Drosophilamelanogaster AAF57833 Plenty of SH3s Drosophila melanogaster NP_523776

Isolated nucleic acids which differ from the nucleotide sequencesencoding a protein designated in Tables 2-4 due to degeneracy in thegenetic code are also within the scope of the invention. For example, anumber of amino acids are designated by more than one triplet. Codonsthat specify the same amino acid, or synonyms (for example, CAU and CACare synonyms for histidine) may result in, “silent” mutations which donot affect the amino acid sequence of the protein. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequences of the subject proteins will exist among mammaliancells. One skilled in the art will appreciate that these variations inone or more nucleotides (up to about 3-5% of the nucleotides) of thenucleic acids encoding a particular protein may exist among individualsof a given species due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of this invention.

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, antisense therapyrefers to administration or in situ generation of oligonucleotide probesor their derivatives which specifically hybridize (e.g. binds) undercellular conditions with the cellular mRNA and/or genomic DNA encodingone of the subject AVMSPs so as to inhibit expression of that protein,e.g. by inhibiting transcription and/or translation. The binding may beby conventional base pair complementarity, or, for example, in the caseof binding to DNA duplexes, through specific interactions in the majorgroove of the double helix. In general, antisense therapy refers to therange of techniques generally employed in the art, and includes anytherapy which relies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes an AVMSP. Alternatively, the antisenseconstruct is an oligonucleotide probe which is generated ex vivo andwhich, when introduced into the cell causes inhibition of expression byhybridizing with the mRNA and/or genomic sequences encoding an AVMSP.Such oligonucleotide probes are preferably modified oligonucleotidewhich are resistant to endogenous nucleases, e.g. exonucleases and/orendonucleases, and is therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat.Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, generalapproaches to constructing oligomers useful in antisense therapy havebeen reviewed, for example, by van der Krol et al., (1988) Biotechniques6:958-976; and Stein et al., (1988) Cancer Res 48:2659-2668.

Accordingly, the modified oligomers of the invention are useful intherapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to, which they specifically bind, such asfor determining the level of expression of a gene of the invention orfor determining whether a gene of the invention contains a geneticlesion.

In another aspect of the invention, the subject nucleic acid is providedin an expression vector comprising a nucleotide sequence encoding asubject AVMSP polypeptide and operably linked to at least one regulatorysequence. Operably linked is intended to mean that the nucleotidesequence is linked to a regulatory sequence in a manner which allowsexpression of the nucleotide sequence. Regulatory sequences areart-recognized and are selected to direct expression of the polypeptidehaving an activity of an AVMSP. Accordingly, the term regulatorysequence includes promoters, enhancers and other expression controlelements. Exemplary regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego,Calif. (1990). For instance, any of a wide variety of expression controlsequences that control the expression of a DNA sequence when operativelylinked to it may be used in these vectors to express DNA sequencesencoding an AVMSP. Such useful expression control sequences, include,for example, the early and late promoters of SV40, tet promoter,adenovirus or cytomegalovirus immediate early promoter, the lac system,the trp system, the TAC or TRC system, T7 promoter whose expression isdirected by T7 RNA polymerase, the major operator and promoter regionsof phage lambda, the control regions for fd coat protein, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other protein encoded by the vector, such as antibiotic markers,should also be considered.

As will be apparent, the subject gene constructs can be used to causeexpression of the subject AVMSP polypeptides in cells propagated inculture, e.g. to produce proteins or polypeptides, including fusionproteins or polypeptides, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject AVMSP. The host cell may be any prokaryotic or eukaryotic cell.For example, a polypeptide of the present invention may be expressed inbacterial cells such as E. coli, insect cells (e.g., using a baculovirusexpression system), yeast, or mammalian cells. Other suitable host cellsare known to those skilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject AVMSP polypeptides. For example, a host celltransfected with an expression vector encoding an AVMSP polypeptide canbe cultured under appropriate conditions to allow expression of thepolypeptide to occur. The polypeptide may be secreted and isolated froma mixture of cells and medium containing the polypeptide. Alternatively,the polypeptide may be retained cytoplasmically and the cells harvested,lysed and the protein isolated. A cell culture includes host cells,media and other byproducts. Suitable media for cell culture are wellknown in the art. The polypeptide can be isolated from cell culturemedium, host cells, or both using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the polypeptide. In a preferred embodiment, the AVMSP is afusion protein containing a domain which facilitates its purification,such as an AVMSP-GST fusion protein, AVMSP-cellulose binding domainfusion protein, etc.

A nucleotide sequence encoding an AVMSP can be used to produce arecombinant form of the protein via microbial or eukaryotic cellularprocesses. Ligating the polynucleotide sequence into a gene construct,such as an expression vector, and transforming or transfecting intohosts, either eukaryotic (yeast, avian, insect or mammalian) orprokaryotic (bacterial) cells, are standard procedures.

A recombinant AVMSP can be produced by ligating the cloned gene, or aportion thereof, into a vector suitable for expression in eitherprokaryotic cells, eukaryotic cells, or both. Expression vehicles forproduction of a recombinant AVMSP include plasmids and other vectors.For instance, suitable vectors for the expression of an AVMSP includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al., (1983)in Experimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

The preferred mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Examplesof other viral (including retroviral) expression systems can be foundbelow in the description of gene therapy delivery systems. The variousmethods employed in the preparation of the plasmids and transformationof host organisms are well known in the art. For other suitableexpression systems for both prokaryotic and eukaryotic cells, as well asgeneral recombinant procedures, see Molecular Cloning A LaboratoryManual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, itmay be desirable to express the recombinant AVMSP by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

It is well known in the art that a methionine at the N-terminal positioncan be enzymatically cleaved by the use of the enzyme methionineaminopeptidase (MAP). MAP has been cloned from E. coli (Ben-Bassat etal., (1987) J. Bacteriol. 169:751-757) and Salmonella typhimurium andits in vitro activity has been demonstrated on recombinant proteins(Miller et al., (1987) PNAS USA 84:2718-1722). Therefore, removal of anN-terminal methionine, if desired, can be achieved either in vivo byexpressing such recombinant polypeptides in a host which produces MAP(e.g., E. coli or CM89 or S. cerevisiae), or in vitro by use of purifiedMAP (e.g., procedure of Miller et al.).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene including a nucleotide sequenceencoding a different polypeptide. This type of expression system can beuseful under conditions where it is desirable, e.g., to produce animmunogenic fragment of an AVMSP. For example, the VP6 capsid protein ofrotavirus can be used as an immunologic carrier protein for portions ofpolypeptide, either in the monomeric form or in the form of a viralparticle. The nucleic acid sequences corresponding to the portion of theAVMSP to which antibodies are to be raised can be incorporated into afusion gene construct which includes coding sequences for a latevaccinia virus structural protein to produce a set of recombinantviruses expressing fusion proteins comprising a portion of the proteinas part of the virion. The Hepatitis B surface antigen can also beutilized in this role as well. Similarly, chimeric constructs coding forfusion proteins containing a portion of an AVMSP and the polioviruscapsid protein can be created to enhance immunogenicity (see, forexample, EP Publication NO: 0259149; and Evans et al., (1989) Nature339:385; Huang et al., (1988) J. Virol. 62:3855; and Schlienger et al.,(1992) J. Virol. 66:2).

The Multiple Antigen Peptide system for peptide-based immunization canbe utilized, wherein a desired portion of an AVMSP is obtained directlyfrom organo-chemical synthesis of the peptide onto an oligomericbranching lysine core (see, for example, Posnett et al., (1988) JBC263:1719 and Nardelli et al., (1992) J. Immunol. 148:914). Antigenicdeterminants of an AVMSP can also be expressed and presented bybacterial cells.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant protein, canallow purification of the expressed fusion protein by affinitychromatography using a Ni²⁺ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified AVMSP (e.g., see Hochuli et al., (1987) J.Chromatography 411:177; and Janknecht et al., PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Exemplary Polypeptides

The present invention also makes available isolated and/or purifiedforms of the subject AVMSPs, which are isolated from, or otherwisesubstantially free of, other intracellular proteins which might normallybe associated with the protein or a particular complex including theprotein. In certain embodiments, polypeptides of the invention have anamino acid sequence that is at least 60% identical to an amino acidsequence as set forth in SEQ ID Nos. 1-39. In other embodiments, thepolypeptide ha an amino acid sequence at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence asset forth in SEQ ID Nos. 1-39.

In another aspect, the invention provides polypeptides that are agonistsor antagonists of AVMSPs. Variants and fragments of an AVMSP may have ahyperactive or constitutive activity, or, alternatively, act to preventAVMSPs from performing one or more functions. For example, a truncatedform lacking one or more domain may have a dominant negative effect.

Another aspect of the invention relates to polypeptides derived from afull-length AVMSP. Isolated peptidyl portions of the subject proteinscan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding suchpolypeptides. In addition, fragments can be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-Moc or t-Boc chemistry. For example, any one of the subject proteinscan be arbitrarily divided into fragments of desired length with nooverlap of the fragments, or preferably divided into overlappingfragments of a desired length. The fragments can be produced(recombinantly or by chemical synthesis) and tested to identity thosepeptidyl fragments which can function as either agonists or antagonistsof the formation of a specific protein complex, or more generally of anAVMSP, such as by microinjection assays.

It is also possible to modify the structure of the subject AVMSPs forsuch purposes as enhancing therapeutic or prophylactic efficacy, orstability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified polypeptides, when designed toretain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the AVMSPs describedin more detail herein. Such modified polypeptides can be produced, forinstance, by amino acid substitution, deletion, or addition.

For instance, it is reasonable to expect, for example, that an isolatedreplacement of a leucine with an isoleucine or valine, an aspartate witha glutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid (i.e. conservativemutations) will not have a major effect on the biological activity ofthe resulting molecule. Conservative replacements are those that takeplace within a family of amino-acids that are related in their sidechains. Genetically encoded amino acids are can be divided into fourfamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids. In similar fashion, the amino acid repertoirecan be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine,isoleucine, serine, threonine, with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine,tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methionine. (see, for example,Biochemistry, 2nd ed., Ed. by L. Stryer, W.H. Freeman and Co., 1981).Whether a change in the amino acid sequence of a polypeptide results ina functional homolog can be readily determined by assessing the abilityof the variant polypeptide to produce a response in cells in a fashionsimilar to the wild-type protein. For instance, such variant forms of anAVMSP can be assessed, e.g., for their ability to bind to anotherpolypeptide, e.g., another AVMSP or another protein involved in viralmaturation. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the subject AVMSPs, as well as truncationmutants, and is especially useful for identifying potential variantsequences (e.g. homologs) that are functional in binding to an AVMSP.The purpose of screening such combinatorial libraries is to generate,for example, AVMSP homologs which can act as either agonists orantagonist, or alternatively, which possess novel activities alltogether. Combinatorially-derived homologs can be generated which have aselective potency relative to a naturally occurring AVMSP. Suchproteins, when expressed from recombinant DNA constructs, can be used ingene therapy protocols.

Likewise, mutagenesis can give rise to homologs which have intracellularhalf-lives dramatically different than the corresponding wild-typeprotein. For example, the altered protein can be rendered either morestable or less stable to proteolytic degradation or other cellularprocess which result in destruction of, or otherwise inactivation of theAVMSP of interest. Such homologs, and the genes which encode them, canbe utilized to alter AVMSP expression by modulating the half-life of theprotein. For instance, a short half-life can give rise to more transientbiological effects and, when part of an inducible expression system, canallow tighter control of recombinant AVMSP levels within the cell. Asabove, such proteins, and particularly their recombinant nucleic acidconstructs, can be used in gene therapy protocols.

In similar fashion, AVMSP homologs can be generated by the presentcombinatorial approach to act as antagonists, in that they are able tointerfere with the ability of the corresponding wild-type protein tofunction.

In a representative embodiment of this method, the amino acid sequencesfor a population of AVMSP homologs are aligned, preferably to promotethe highest homology possible. Such a population of variants caninclude, for example, homologs from one or more species, or homologsfrom the same species but which differ due to mutation. Amino acidswhich appear at each position of the aligned sequences are selected tocreate a degenerate set of combinatorial sequences. In a preferredembodiment, the combinatorial library is produced by way of a degeneratelibrary of genes encoding a library of polypeptides which each includeat least a portion of potential AVMSP sequences. For instance, a mixtureof synthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential AVMSP nucleotidesequences are expressible as individual polypeptides, or alternatively,as a set of larger fusion proteins (e.g. for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then be ligated into an appropriategene for expression. The purpose of a degenerate set of genes is toprovide, in one mixture, all of the sequences encoding the desired setof potential AVMSP sequences. The synthesis of degenerateoligonucleotides is well known in the art (see for example, Narang, SA(1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc.3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevierpp 273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakuraet al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res.11:477). Such techniques have been employed in the directed evolution ofother proteins (see, for example, Scott et al., (1990) Science249:386-390; Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin etal., (1990) Science 249: 404-406; Cwirla et al., (1990) PNAS USA 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, AVMSP homologs (both agonist andantagonist forms) can be generated and isolated from a library byscreening using, for example, alanine scanning mutagenesis and the like(Ruf et al., (1994) Biochemistry 33:1565-1572; Wang et al., (1994) J.Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-118;Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et al.,(1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry30:10832-10838; and Cunningham et al., (1989) Science 244:1081-1085), bylinker scanning mutagenesis (Gustin et al., (1993) Virology 193:653-660;Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al.,(1982) Science 232:316); by saturation mutagenesis (Meyers et al.,(1986) Science 232:613); by PCR mutagenesis (Leung et al., (1989) MethodCell Mol Biol 1:11-19); or by random mutagenesis, including chemicalmutagenesis, etc. (Miller et al., (1992) A Short Course in BacterialGenetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al.,(1994) Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis,particularly in a combinatorial setting, is an attractive method foridentifying truncated (bioactive) forms of AVMSPs.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of AVMSP homologs. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

In an illustrative embodiment of a screening assay, candidatecombinatorial gene products of one of the subject proteins are displayedon the surface of a cell or virus, and the ability of particular cellsor viral particles to bind an AVMSP, eg. a protein designated in Tables2 and 3, is detected in a “panning assay”. For instance, a library ofAVMSP-IP variants can be cloned into the gene for a surface membraneprotein of a bacterial cell (Ladner et al., WO 88/06630; Fuchs et al.,(1991) Bio/Technology 9:1370-1371; and Gowardi et al., (1992) TIBS18:136-140), and the resulting fusion protein detected by panning, e.g.using a fluorescently labeled molecule which binds the AVMSP-IP, such asFITC-labelled AVMSP, to score for potentially functional homologs. Cellscan be visually inspected and separated under a fluorescence microscope,or, where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

In similar fashion, the gene library can be expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, a large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd, and f1 are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al., PCT publication WO 90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al., (1992) J. Biol. Chem.267:16007-16010; Griffiths et al., (1993) EMBO J. 12:725-734; Clacksonet al., (1991) Nature 352:624-628; and Barbas et al., (1992) PNAS USA89:4457-4461).

The invention also provides for reduction of the subject AVMSPs togenerate mimetics, e.g. peptide or non-peptide agents, which are able tomimic binding of the authentic protein to another cellular partner. Suchmutagenic techniques as described above, as well as the thioredoxinsystem, are also particularly useful for mapping the determinants of anAVMSP which participate in protein-protein interactions involved in, forexample, binding of proteins involved in viral maturation to each other.To illustrate, the critical residues of an AVMSP which are involved inmolecular recognition of a substrate protein can be determined and usedto generate AVMSP-derived peptidomimetics which bind to the substrateprotein, and by inhibiting AVMSP binding, act to inhibit its biologicalactivity. By employing, for example, scanning mutagenesis to map theamino acid residues of an AVMSP which are involved in binding to anotherpolypeptide, peptidomimetic compounds can be generated which mimic thoseresidues involved in binding. For instance, non-hydrolyzable peptideanalogs of such resides can be generated using benzodiazepine (e.g., seeFreidinger et al., in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al., in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988); substituted gamma lactamrings (Garvey et al., in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al., (1986) J. Med. Chem. 29:295; and Ewensonet al., in Peptides: Structure and Function (Proceedings of the 9thAmerican Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985),b-turn dipeptide cores (Nagai et al., (1985) Tetrahedron Lett 26:647;and Sato et al., (1986) J Chem Soc. Perlin Trans 1:1231), andb-aminoalcohols (Gordon et al., (1985) Biochem Biophys Res Commun126:419; and Dann et al., (1986) Biochem Biophys Res Commun 134:71).

5. Antibodies and Uses Therefor

Another aspect of the invention pertains to an antibody specificallyreactive with an AVMSP, e.g., a wild-type or mutated AVMSP. For example,by using immunogens derived from an AVMSP, e.g., based on the cDNAsequences, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (See, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the peptide (e.g., a mammalian AVMSP or anantigenic fragment which is capable of eliciting an antibody response,or a fusion protein as described above). Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques well known in the art. An immunogenic portion of anAVMSP can be administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassays can be used with theimmunogen as antigen to assess the levels of antibodies. In a preferredembodiment, the subject antibodies are immunospecific for antigenicdeterminants of an AVMSP of a mammal, e.g., antigenic determinants of aprotein set forth in SEQ ID Nos: 1-39.

In one embodiment, antibodies are specific for a RING domain, an SH3domain, an SH2 domain, and a C2 domain, and preferably the domain ispart of an AVMSP, such as a domain of an AVMSP shown in one of SEQ IDNOs: 1-39. In another embodiment, the antibodies are immunoreactive withone or more proteins having an amino acid sequence that is at least 80%identical to an amino acid sequence as set forth in SEQ ID Nos. 1-39. Inother embodiment, an antibody is immunoreactive with one or moreproteins having an amino acid sequence that is 85%, 90%, 95%, 98%, 99%or identical to an amino acid sequence as set forth in SEQ ID Nos. 1-39.

In a further embodiment, an antibody of the invention disrupts thedirect or indirect interaction between an AVMSP polypeptide and anAVMSP-AP.

Following immunization of an animal with an antigenic preparation of anAVMSP, anti-AVMSP antisera can be obtained and, if desired, polyclonalanti-AVMSP antibodies isolated from the serum. To produce monoclonalantibodies, antibody-producing cells (lymphocytes) can be harvested froman immunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, and include, for example, thehybridoma technique (originally developed by Kohler and Milstein, (1975)Nature, 256: 495-497), the human B cell hybridoma technique (Kozbar etal., (1983) Immunology Today, 4: 72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., (1985) MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with a mammalian AVMSP polypeptide of the presentinvention and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells. In one embodiment anti-human AVMSP antibodiesspecifically react with the protein encoded by a nucleic acid having SEQID Nos 40-79.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectmammalian AVMSP polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example, F(ab)₂fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific, single-chain, and chimeric and humanizedmolecules having affinity for an AVMSP protein conferred by at least oneCDR region of the antibody. In preferred embodiments, the antibodies,the antibody further comprises a label attached thereto and able to bedetected, (e.g., the label can be a radioisotope, fluorescent compound,enzyme or enzyme co-factor).

Anti-AVMSP antibodies can be used, e.g., to monitor AVMSP levels in anindividual, particularly the presence of AVMSPs in the plasma membranefor determining whether or not said patient is infected with a virussuch as an RNA virus, or allowing determination of the efficacy of agiven treatment regimen for an individual afflicted with such adisorder. In addition, AVMSPs are understood to localize, occasionally,to the released viral particle. Viral particles may be collected andassayed for the presence of one or more AVMSPs. The level of AVMSP maybe measured in a variety of sample types such as, for example, cellsand/or in bodily fluid, such as in blood samples.

Another application of anti-AVMSP antibodies of the present invention isin the immunological screening of cDNA libraries constructed inexpression vectors such as gt11, gt18-23, ZAP, and ORF8. Messengerlibraries of this type, having coding sequences inserted in the correctreading frame and orientation, can produce fusion proteins. Forinstance, gt11 will produce fusion proteins whose amino termini consistof β-galactosidase amino acid sequences and whose carboxy terminiconsist of a foreign polypeptide. Antigenic epitopes of an AVMSP, e.g.,other orthologs of a particular protein or other paralogs from the samespecies, can then be detected with antibodies, as, for example, reactingnitrocellulose filters lifted from infected plates with the appropriateanti-AVMSP antibodies. Positive phage detected by this assay can then beisolated from the infected plate. Thus, the presence of AVMSP homologscan be detected and cloned from other animals, as can alternate isoforms(including splice variants) from humans.

6. Homology Searching of Nucleotide and Polypeptide Sequences

The nucleotide or amino acid sequences of the invention may be used asquery sequences against databases such as GenBank, SwissProt, BLOCKS,and Pima II. These databases contain previously identified and annotatedsequences that can be searched for regions of homology (similarity)using BLAST, which stands for Basic Local Alignment Search Tool(Altschul S F (1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) JMol Biol 215:403-10).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith, R. F. and T. F. Smith (1992; ProteinEngineering 5:35-51), incorporated herein by reference, can be used whendealing with primary sequence patterns and secondary structure gappenalties. As disclosed in this application, sequences have lengths ofat least 49 nucleotides and no more than 12% uncalled bases (where N isrecorded rather than A, C, G, or T).

The BLAST approach, as detailed in Karlin and Altschul (1993; Proc NatAcad Sci 90:5873-7) and incorporated herein by reference, searchesmatches between a query sequence and a database sequence, to evaluatethe statistical significance of any matches found, and to report onlythose matches which satisfy the user-selected threshold of significance.Preferably the threshold is set at 10-25 for nucleotides and 3-15 forpeptides.

7. Diagnostic Assays

A further aspect of the invention includes diagnostic assays fordetermining whether a cell is infected with a virus and forcharacterizing the nature, progression and/or infectivity of theinfection.

In one embodiment, it is contemplated that AVMSPs certain associatedproteins localize to different regions of the cell depending on thefunction being performed. In the course of normal activities, it isexpected that AVMSPs will be free in the cytoplasm or associated with anintracellular organelle, such as the nucleus, the Golgi network, etc.During a viral infection, certain AVMSPs are recruited to the cellmembrane to participate in viral maturation, including ubiquitinationand membrane fusion. As a result, the detection of an AVMSP associatedwith the plasma membrane fraction is indicative of a viral infection.Additionally, the presence of AVMSPs at the plasma membrane wouldsuggest that the infective virus is in the process of reproducing and istherefore actively engaged in infective or lytic activity (versus alysogenic or otherwise dormant state).

Association of the proteins of the invention with the plasma membranemay be detected using a variety of techniques known in the art. Forexample, membrane preparations may be prepared by breaking open thecells (via sonication or detergent lysis) and then separating themembrane components from the cytosolic fraction via centrifugation.Segregation of proteins into the membrane fraction can be detected withantibodies specific for the protein of interest, for example by usingWestern blot analysis or ELISA techniques. Plasma membranes may beseparated from intracellular membranes on the basis of density usingdensity gradient centrifugation. Alternatively, plasma membranes may beobtained by chemically or enzymatically modifying the surface of thecell and affinity purifying the plasma membrane by selectively bindingthe modifications. An exemplary modification includes non-specificbiotinylation of proteins at the cell surface. Plasma membranes may alsobe selected for by affinity purifying for abundant plasma membraneproteins.

Transmembrane AVMSP proteins containing an extracellular domain can bedetected using FACS analysis. For FACS analysis, whole cells areincubated with a fluorescently labeled antibody (e.g., an FITC-labelledantibody) capable of recognizing the extracellular domain of the proteinof interest. The level of fluorescent staining of the cells may then bedetermined by FACS analyses (see e.g., Weiss and Stobo, (1984) J. Exp.Med., 160:1284-1299). Such proteins are expected to reside onintracellular membranes in uninfected cells and the plasma membrane ininfected cells. FACS analysis would fail to detect an extracellulardomain unless the protein is present at the plasma membrane.

In a further embodiment, proteins associated with the membranes of cellsand/or viral particles may be profiled. Profiling involves identifyingthe presence or absence of more than one protein in the membraneassociated fraction of a sample. For example, the presence of AVMSPs aredetected in the membrane associated fraction of cells obtained from aperson suspected of a viral infection. Similar profiles may be developedfrom subjects infected by known viruses or subjects thought to be freeof infection. Profiles may be compared to identify proteins that changein abundance, or qualitatively (eg. in terms of PL molecular weight, orother indicators of post-translational modification). Profiles may becompiled into a database for computer-assisted comparisons. Comparisonof profiles may be used to identify an AVMSP that is altered in responseto a certain viral infection. This AVMSP may then be used as adiagnostic for that type of viral infection. The AVMSP may also then beused as a target to identify therapeutic agents that will interfere withits function in the infection. Exemplary profiles of the invention willinclude information about the abundance of more than one AVMSP selectedfrom those represented by SEQ ID Nos. 1-39. Other exemplary profileswill include information about the abundance of 5, 10, 20, 30, 40, 50,60 or all of the proteins represented by SEQ ID Nos.1-39.

Localization of the proteins of the invention may also be determinedusing histochemical techniques. For example, cells may be fixed andstained with a fluorescently labeled antibody specific for the proteinof interest. The stained cells may then be examined under the microscopeto determine the subcellular localization of the antibody boundproteins.

In addition, as noted above, AVMSPs may localize to released or buddingviral particles. The presence of these proteins in viral particles maybe determined by a variety of methods. For example, viral particles maybe enriched and analyzed by Western blot or ELISA. As another example,viral particles or cells having budding viroids ay be examined byelectron microscopy. Immunogold labeling, for example, is useful forlocalizing AVMSPs by electron microscopy.

Samples to be used for diagnostic assays may include essentially anysample comprising cells and/or viral particles or a sample prepared froma cellular sample. Exemplary samples would include fluid samples (eg.blood, urine, saliva, mucus, broncheoalveolar lavage, cerebrospinalfluid etc.). Other fluids comprising cells and/or viral particles arewell known to those of skill in the art. Other sample types includestool samples, tissue biopsies and any processed or purified form of theabove.

8. Drug Screening Assays

The present invention also provides assays for identifying therapeuticswhich either interfere with or promote viral maturation, particularly byaffecting AVMSP function. In one embodiment, the assay detects agentswhich inhibit interaction of one or more subject AVMSPs with anAVMSP-AP. In another embodiment, the assay detects agents which modulatethe intrinsic biological activity of an AVMSP, AVMSP complex, such as anenzymatic activity, binding to other cellular components, cellularcompartmentalization, and the like. Such modulators can be used, forexample, in the treatment of viral infections and/or particularly viralinfections by a virus that uses a Gag-dependent maturation system (eg.retrovirus, rhabdovirus, filovirus).

In one aspect, the invention provides methods and compositions for theidentification of compositions that interfere with the function ofAVMSPs. Given the critical role of AVMSPs in virion release,compositions that perturb the formation or stability of theprotein-protein interactions between AVMSPs and the proteins that theyinteract with, such as AVMSP-APs, are candidate pharmaceuticals for thetreatment of viral infections.

While not wishing to be bound to mechanism, it is postulated that AVMSPspromote the assembly of protein complexes that are critically importantin release of virions. Complexes of the invention may include acombination of at least one of the following: a polypeptide comprisingan AVMSP, a Gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60,Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme,tsg101, a cullin, AP-1, AP-2, and a clathrin.

The type of complex formed by an AVMSP will depend upon the domainspresent in the protein. While not intended to be limiting, exemplarydomains of potential interacting proteins are provided below. An SH3domain is expected to interact with one or more small GTPases, such asmembers of the Arf, Rab, Rac and Rho families. In addition, thefollowing Table provides a list of exemplary protein domains that areassociated with the formation of AVMSP complexes: TABLE 5 Domain NameInteracting motif Description 1 SH2 Yxxφ The Src homology 2 (SH2) domainis a protein domain of about 100 amino-acid residues first identified asa conserved sequence region between the oncoproteins Src and Fps.Similar sequences were later found in many other intracellularsignal-transducing proteins. SH2 domains function as regulatory modulesof intracellular signalling cascades by interacting with high affinityto phosphotyrosine-containing target peptides in a sequence-specific andstrictly phosphorylation-dependent manner. 2 SH3 PxRPxR (proline The Srchomology 3 (SH3) domain is a small protein rich) domain of about 60amino-acid residues first identified as a conserved sequence in thenon-catalytic part of several cytoplasmic protein tyrosine kinases (e.g.Src, Abl, Lck). Since then, it has been found in a great variety ofother intracellular or membrane-associated proteins. The function of theSH3 domain is to mediate assembly of specific protein complexes viabinding to proline-rich peptides. 3 Endocytosis Yxxφ Tyrosine notphosphorylated motifs: (D/E)xxxLL μ2 subunit of (dileucine) AP2 FYRALβadaptin or NPXY through an adaptor to AP2 (NEF) AP2? Clatherin 4Clatherin Multiple DLL or AP-2, AP180, AP1 assembly SLL domain 5 C2Ca2+, Ca2+-binding motif present in phospholipases, proteinphospholipids, kinases C, and synaptotamins (among others). Some doinositol not appear to contain Ca2+-binding sites. Particular C2spolyphosphates. appear to bind phospholipids, inositol polyphosphates,and intracellular proteins. Unusual occurrence in perforin.Synaptotagmin and PLC C2s are permuted in sequence with respect to N-and C-terminal beta strands. 6 WW [AP]-P-P-[AP]-Y Found in dystrophin.The domain, which spans about 35 residues, is repeated up to 4 times insome proteins. It has been shown to bind proteins with particularproline-motifs, and thus resembles somewhat SH3 domains. The name WW orWWP derives from the presence of Trp as well as that of a conserved Pro.It is frequently associated with other domains typical for proteins insignal transduction processes. 7 RCC1 Ran GTPase The regulator ofchromosome condensation (RCC1) is a eukaryotic protein, which binds tochromatin and interacts with ran, a nuclear GTP-binding protein topromote the loss of bound GDP and the uptake of fresh GTP, thus actingas a guanine-nucleotide dissociation stimulator (GDS).

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. Assay formats whichapproximate such conditions as formation of protein complexes, enzymaticactivity, and even an AVMSP-mediated membrane reorganization activity,can be generated in many different forms, and include assays based oncell-free systems, e.g. purified proteins or cell lysates, as well ascell-based assays which utilize intact cells. Simple binding assays canalso be used to detect agents which, by disrupting the binding of AVMSPsto interacting protein, or the binding of an AVMSP or complex to asubstrate, can inhibit viral maturation. Agents to be tested for theirability to act as viral maturation inhibitors can be produced, forexample, by bacteria, yeast or other organisms (e.g. natural products),produced chemically (e.g. small molecules, including peptidomiimetics),or produced recombinantly. In a preferred embodiment, the test agent isa small organic molecule, e.g., other than a peptide or oligonucleotide,having a molecular weight of less than about 2,000 daltons.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays of the present invention which are performed in cell-freesystems, such as may be developed with purified or semi-purifiedproteins or with lysates, are often preferred as “primary” screens inthat they can be generated to permit rapid development and relativelyeasy detection of an alteration in a molecular target which is mediatedby a test compound. Moreover, the effects of cellular toxicity and/orbioavailability of the test compound can be generally ignored in the invitro system, the assay instead being focused primarily on the effect ofthe drug on the molecular target as may be manifest in an alteration ofbinding affinity with other proteins or changes in enzymatic propertiesof the molecular target.

In preferred in vitro embodiments of the present assay, a reconstitutedAVMSP complex comprises a reconstituted mixture of at leastsemi-purified proteins. By semi-purified, it is meant that the proteinsutilized in the reconstituted mixture have been previously separatedfrom other cellular or viral proteins. For instance, in contrast to celllysates, the proteins involved in AVMSP complex formation, are presentin the mixture to at least 50% purity relative to all other proteins inthe mixture, and more preferably are present at 90-95% purity. Incertain embodiments of the subject method, the reconstituted proteinmixture is derived by mixing highly purified proteins such that thereconstituted mixture substantially lacks other proteins (such as ofcellular or viral origin) which might interfere with or otherwise alterthe ability to measure AVMSP complex assembly and/or disassembly.

Assaying AVMSP complexes, in the presence and absence of a candidateinhibitor, can be accomplished in any vessel suitable for containing thereactants. Examples include microtitre plates, test tubes, andmicro-centrifuge tubes.

In one embodiment of the present invention, drug screening assays can begenerated which detect inhibitory agents on the basis of their abilityto interfere with assembly or stability of the AVMSP complex. In anexemplary binding assay, the compound of interest is contacted with amixture comprising an AVMSP polypeptide and is at least one interactingpolypeptide. Detection and quantification of AVMSP complexes provides ameans for determining the compound's efficacy at inhibiting (orpotentiating) interaction between the two polypeptides. The efficacy ofthe compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. In the control assay, the formation of complexes isquantitated in the absence of the test compound.

Complex formation between the AVMSP polypeptides or between an AVMSP anda substrate polypeptide may be detected by a variety of techniques, manyof which are effectively described above. For instance, modulation inthe formation of complexes can be quantitated using, for example,detectably labeled proteins (e.g. radiolabeled, fluorescently labeled,or enzymatically labeled), by immunoassay, or by chromatographicdetection. Surface plasmon resonance systems, such as those availablefrom BioCore, Inc., may also be used to detect protein-proteininteraction Often, it will be desirable to immobilize one of thepolypeptides to facilitate separation of complexes from uncomplexedforms of one of the proteins, as well as to accommodate automation ofthe assay. In an illustrative embodiment, a fusion protein can beprovided which adds a domain that permits the protein to be bound to aninsoluble matrix. For example, GST-AVMSP or -AMVSP fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with a potential interacting protein, e.g. an ³⁵S-labeledpolypeptide, and the test compound and incubated under conditionsconducive to complex formation. Following incubation, the beads arewashed to remove any unbound interacting protein, and the matrixbead-bound radiolabel determined directly (e.g. beads placed inscintillant), or in the supernatant after the complexes are dissociated,e.g. when microtitre plate is used. Alternatively, after washing awayunbound protein, the complexes can be dissociated from the matrix,separated by SDS-PAGE gel, and the level of interacting polypeptidefound in the matrix-bound fraction quantitated from the gel usingstandard electrophoretic techniques.

In yet another embodiment, the AVMSP and potential interactingpolypeptide can be used to generate an interaction trap assay (see also,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696), for subsequently detecting agents which disrupt binding ofthe proteins to one and other.

In particular, the method makes use of chimeric genes which expresshybrid proteins. To illustrate, a first hybrid gene comprises the codingsequence for a DNA-binding domain of a transcriptional activator can befused in frame to the coding sequence for a “bait” protein, e.g., anAVMSP polypeptide of sufficient length to bind to a potentialinteracting protein. The second hybrid protein encodes a transcriptionalactivation domain fused in frame to a gene encoding a “fish” protein,e.g., a potential interacting protein of sufficient length to interactwith the AVMSP polypeptide portion of the bait fusion protein. If thebait and fish proteins are able to interact, e.g., form an AVMSPcomplex, they bring into close proximity the two domains of thetranscriptional activator. This proximity causes transcription of areporter gene which is operably linked to a transcriptional regulatorysite responsive to the transcriptional activator, and expression of thereporter gene can be detected and used to score for the interaction ofthe bait and fish proteins.

In accordance with the present invention, the method includes providinga host cell, preferably a yeast cell, e.g., Kluyverei lactis,Schizosaccharomyces pombe, Ustilago maydis, Saccharomyces cerevisiae,Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Pichiapastoris, Candida tropicalis, and Hansenula polymorpha, though mostpreferably S cerevisiae or S. pombe. The host cell contains a reportergene having a binding site for the DNA-binding domain of atranscriptional activator used in the bait protein, such that thereporter gene expresses a detectable gene product when the gene istranscriptionally activated. The first chimeric gene may be present in achromosome of the host cell, or as part of an expression vector.Interaction trap assays may also be performed in mammalian and bacterialcell types.

The host cell also contains a first chimeric gene which is capable ofbeing expressed in the host cell. The gene encodes a chimeric protein,which comprises (i) a DNA-binding domain that recognizes the responsiveelement on the reporter gene in the host cell, and (ii) a bait protein,such as an AVMSP polypeptide sequence.

A second chimeric gene is also provided which is capable of beingexpressed in the host cell, and encodes the “fish” fusion protein. Inone embodiment, both the first and the second chimeric genes areintroduced into the host cell in the form of plasmids. Preferably,however, the first chimeric gene is present in a chromosome of the hostcell and the second chimeric gene is introduced into the host cell aspart of a plasmid.

Preferably, the DNA-binding domain of the first hybrid protein and thetranscriptional activation domain of the second hybrid protein arederived from transcriptional activators having separable DNA-binding andtranscriptional activation domains. For instance, these separateDNA-binding and transcriptional activation domains are known to be foundin the yeast GAL4 protein, and are known to be found in the yeast GCN4and ADR1 proteins. Many other proteins involved in transcription alsohave separable binding and transcriptional activation domains which makethem useful for the present invention, and include, for example, theLexA and VP16 proteins. It will be understood that other (substantially)transcriptionally-inert DNA-binding domains may be used in the subjectconstructs; such as domains of ACE1, lcI, lac repressor, jun or fos. Inanother embodiment, the DNA-binding domain and the transcriptionalactivation domain may be from different proteins. The use of a LexA DNAbinding domain provides certain advantages. For example, in yeast, theLexA moiety contains no activation function and has no known effect ontranscription of yeast genes. In addition, use of LexA allows controlover the sensitivity of the assay to the level of interaction (see, forexample, the Brent et al. PCT publication WO94/10300).

In preferred embodiments, any enzymatic activity associated with thebait or fish proteins is inactivated, e.g., dominant negative or othermutants of an AVMSP can be used.

Continuing with the illustrated example, the AVMSP-mediated interaction,if any, between the bait and fish fusion proteins in the host cell,therefore, causes the activation domain to activate transcription of thereporter gene. The method is carried out by introducing the firstchimeric gene and the second chimeric gene into the host cell, andsubjecting that cell to conditions under which the bait and fish fusionproteins and are expressed in sufficient quantity for the reporter geneto be activated. The formation of an AVMSP/interacting protein complexresults in a detectable signal produced by the expression of thereporter gene. Accordingly, the level of formation of a complex in thepresence of a test compound and in the absence of the test compound canbe evaluated by detecting the level of expression of the reporter genein each case. Various reporter constructs may be used in accord with themethods of the invention and include, for example, reporter genes whichproduce such detectable signals as selected from the group consisting ofan enzymatic signal, a fluorescent signal, a phosphorescent signal anddrug resistance.

One aspect of the present invention provides reconstituted proteinpreparations including an AVMSP and one or more interactingpolypeptides.

In still further embodiments of the present assay, the AVMSP complex isgenerated in whole cells, taking advantage of cell culture techniques tosupport the subject assay. For example, as described below, the AVMSPcomplex can be constituted in a eukaryotic cell culture system,including mammalian and yeast cells. Often it will be desirable toexpress one or more viral proteins (eg. Gag or Env) in such a cell alongwith a subject AVMSP. It may also be desirable to infect the cell with avirus of interest. Advantages to generating the subject assay in anintact cell include the ability to detect inhibitors which arefunctional in an environment more closely approximating that whichtherapeutic use of the inhibitor would require, including the ability ofthe agent to gain entry into the cell. Furthermore, certain of the invivo embodiments of the assay, such as examples given below, areamenable to high through-put analysis of candidate agents.

The components of the AVMSP can be endogenous to the cell selected tosupport the assay. Alternatively, some or all of the components can bederived from exogenous sources. For instance, fusion proteins can beintroduced into the cell by recombinant techniques (such as through theuse of an expression vector), as well as by microinjecting the fusionprotein itself or mRNA encoding the fusion protein.

In any case, the cell is ultimately manipulated after incubation with acandidate drug and assayed for an AVMSP activity. AVMSP activities mayinclude, without limitation, complex formation, ubiquitination andmembrane fusion events (eg. release of viral buds or fusion ofvesicles). AVMSP complex formation may be assessed byimmunoprecipitation and analysis of co-immunoprecipiated proteins oraffinity purification and analysis of co-purified proteins. FluorescenceResonance Energy Transfer (FRET)-based assays may also be used todetermine complex formation. Fluorescent molecules having the properemission and excitation spectra that are brought into close proximitywith one another can exhibit FRET. The fluorescent molecules are chosensuch that the emission spectrum of one of the molecules (the donormolecule) overlaps with the excitation spectrum of the other molecule(the acceptor molecule). The donor molecule is excited by light ofappropriate intensity within the donor's excitation spectrum. The donorthen emits the absorbed energy as fluorescent light. The fluorescentenergy it produces is quenched by the acceptor molecule. FRET can bemanifested as a reduction in the intensity of the fluorescent signalfrom the donor, reduction in the lifetime of its excited state, and/orre-emission of fluorescent light at the longer wavelengths (lowerenergies) characteristic of the acceptor. When the fluorescent proteinsphysically separate, FRET effects are diminished or eliminated. (U.S.Pat. No. 5,981,200).

For example, a cyan fluorescent protein is excited by light at roughly425-450 nm wavelength and emits light in the range of 450-500 nm. Yellowfluorescent protein is excited by light at roughly 500-525 nm and emitslight at 525-500 nm. If these two proteins are placed in solution, thecyan and yellow fluorescence may be separately visualized. However, ifthese two proteins are forced into close proximity with each other, thefluorescent properties will be altered by FRET. The bluish light emittedby CFP will be absorbed by YFP and re-emitted as yellow light. Thismeans that when the proteins are stimulated with light at wavelength 450nm, the cyan emitted light is greatly reduced and the yellow light,which is not normally stimulated at this wavelength, is greatlyincreased. FRET is typically monitored by measuring the spectrum ofemitted light in response to stimulation with light in the excitationrange of the donor and calculating a ratio between the donor-emittedlight and the acceptor-emitted light. When the donor:acceptor emissionratio is high, FRET is not occurring and the two fluorescent proteinsare not in close proximity. When the donor: acceptor emission ratio islow, FRET is occurring and the two fluorescent proteins are in closeproximity. In this manner, the interaction between a first and secondpolypeptide may be measured.

The occurrence of FRET also causes the fluorescence lifetime of thedonor fluorescent moiety to decrease. This change in fluorescencelifetime can be measured using a technique termed fluorescence lifetimeimaging technology (FLIM) (Verveer et al. (2000) Science 290: 1567-1570;Squire et al. (1999) J. Microsc. 193: 36; Verveer et al. (2000) Biophys.J. 78: 2127). Global analysis techniques for analyzing FLIM data havebeen developed. These algorithms use the understanding that the donorfluorescent moiety exists in only a limited number of states each with adistinct fluorescence lifetime. Quantitative maps of each state can begenerated on a pixel-by-pixel basis.

To perform FRET-based assays, the AVMSP and the interacting protein ofinterest are both fluorescently labeled. Suitable fluorescent labelsare, in view of this specification, well known in the art. Examples areprovided below, but suitable fluorescent labels not specificallydiscussed are also available to those of skill in the art. Fluorescentlabeling may be accomplished by expressing a polypeptide as a fusionprotein with a fluorescent protein, for example fluorescent proteinsisolated from jellyfish, corals and other coelenterates. Exemplaryfluorescent proteins include the many variants of the green fluorescentprotein (GFP) of Aequoria victoria. Variants may be brighter, dimmer, orhave different excitation and/or emission spectra Certain variants arealtered such that they no longer appear green, and may appear blue,cyan, yellow or red (termed BFP, CFP, YFP and RFP, respectively).Fluorescent proteins may be stably attached to polypeptides through avariety of covalent and noncovalent linkages, including, for example,peptide bonds (eg. expression as a fusion protein), chemicalcross-linking and biotin-streptavidin coupling. For examples offluorescent proteins, see U.S. Pat. Nos. 5,625,048; 5,777,079;6,066,476; 6,124,128; Prasher et al. (1992) Gene, 111:229-233; Heim etal. (1994) Proc. Natl. Acad. Sci., USA, 91:12501-04; Ward et al. (1982)Photochem. Photobiol, 35:803-808; Levine et al. (1982) Comp. Biochem.Physiol., 72B:77-85; Tersikh et al. (2000) Science 290: 1585-88.

Other exemplary fluorescent moieties well known in the art includederivatives of fluorescein, benzoxadioazole, coumarin, eosin, LuciferYellow, pyridyloxazole and rhodamine. These and many other exemplaryfluorescent moieties may be found in the Handbook of Fluorescent Probesand Research Chemicals (2000, Molecular Probes, Inc.), along withmethodologies for modifying polypeptides with such moieties. Exemplaryproteins that fluoresce when combined with a fluorescent moiety include,yellow fluorescent protein from Vibrio fischeri (Baldwin et al. (1990)Biochemistry 29:5509-15), peridinin-chlorophyll a binding protein fromthe dinoflagellate Symbiodinium sp. (Morris et al. (1994) PlantMolecular Biology 24:673:77) and phycobiliproteins from marinecyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin(Wilbanks et al. (1993) J. Biol. Chem. 268:1226-35). These proteinsrequire flavins, peridinin-chlorophyll a and various phycobilins,respectively, as fluorescent co-factors.

FRET-based assays may be used in cell-based assays and in cell-freeassays. FRET-based assays are amenable to high-throughput screeningmethods including Fluorescence Activated Cell Sorting and fluorescentscanning of microtiter arrays.

10. Methods and Compositions for Treatment of Viral Disorders

In a further aspect, the invention provides methods and compositions fortreatment of viral disorders, and particularly disorders caused by RNAviruses, including but not limited to retroviruses, rhabdoviruses andfiloviruses. Preferred therapeutics of the invention function bydisrupting the biological activity of an AVMSP or AVMSP complex in viralmaturation.

Exemplary therapeutics of the invention include antisense therapies,RNAi or siRNA therapies, polypeptides, peptidomimetics, antibodies andsmall molecules.

Antisense therapies of the invention include methods of introducingantisense nucleic acids to disrupt the expression of AVMSPs or proteinsthat are necessary for AVMSP function. RNAi or siRNA therapies bothrefer to the administration of short double stranded RNA molecules thatare complementary to a portion of an AVMSP transcript to achieve adecrease in the expression of that AVMSP and, thereby, a decrease in thefunction of that AVMSP. RNA molecules for siRNA may be prepared as atherapeutic composition by, for example, mixing them with a suitablecarrier, such as a lipid or polycationic carrier that facilitatesdelivery of the nucleic acid across the membrane of the targeted cells.

Therapeutic polypeptides may be generated by designing polypeptides tomimic certain protein domains important in the formation of AVMSPcomplexes. For example, a polypeptide comprising an SH3 domain willcompete for binding to an SH3 domain and will therefore act to disruptbinding of a Gag protein, for example, to the AVMSP complex. Likewise, apolypeptide that resembles an L domain may disrupt recruitment of Gag tothe AVMSP complex. Such polypeptide mimetics may be targeted to any of avariety of domains, including for example, those domains listed in Table5.

In view of the specification, methods for generating antibodies directedto epitopes of AVMSPs and AVMSP-interacting proteins are known in theart. Antibodies may be introduced into cells by a variety of methods.One exemplary method comprises generating a nucleic acid encoding asingle chain antibody that is capable of disrupting an AVMSP complex.Such a nucleic acid may be conjugated to antibody that binds toreceptors on the surface of target cells. It is contemplated that incertain embodiments, the antibody may target viral proteins that arepresent on the surface of infected cells, and in this way deliver thenucleic acid only to infected cells. Once bound to the target cellsurface, the antibody is taken up by endocytosis, and the conjugatednucleic acid is transcribed and translated to produce a single chainantibody that interacts with and disrupts the targeted AVMSP complex.Nucleic acids expressing the desired single chain antibody may also beintroduced into cells using a variety of more conventional techniques,such as viral transfection (eg. using an adenoviral system) orliposome-mediated transfection.

Small molecules of the invention may be identified for their ability tomodulate the formation of AVMSP complexes, as described above.

In view of the teachings herein, one of skill in the art will understandthat the methods and compositions of the invention are applicable to awide range of RNA viruses including retroviruses. While not intended tobe limiting, relevant retroviruses include: C-type retrovirus whichcauses lymphosarcoma in Northern Pike, the C-type retrovirus whichinfects mink, the caprine lentivirus which infects sheep, the EquineInfectious Anemia Virus EIAV, the C-type retrovirus which infects pigs,the Avian Leukosis Sarcoma Virus (ALSV), the Feline Leukemia Virus(FeLV), the Feline Aids Virus, the Bovine Leukemia Virus (BLV), theSimian Leukemia Virus (SLV), the Simian Immuno-deficiency Virus (SIV),the Human T-cell Leukemia Virus type-I (HTLV-I), the Human T-cellLeukemia Virus type-II (HTLV-I), Human Immunodeficiency virus type-2(HIV-2) and Human Immunodeficiency virus type-1 (HIV-1). Other RNAviruses include picornaviruses such as enterovirus, poliovirus,coxsackievirus and hepatitis A virus, the caliciviruses, includingNorwalk-like viruses, the rhabdoviruses, including rabies virus, thetogaviruses including alphaviruses, Semliki Forest virus, denguevirus,yellow fever virus and rubella virus, the orthomyxoviruses, includingType A, B, and C influenza viruses, the bunyaviruses, including the RiftValley fever virus and the hantavirus, the filoviruses such as Ebolavirus and Marburg virus, and the paramyxoviruses, including mumps virusand measles virus.

11. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining The Ld₅₀ (The Dose Lethal To 50% Of ThePopulation) And The Ed₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic induces are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

12. Formulation and Use

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ation oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated, inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

For therapies involving the administration of nucleic acids, theoligomers of the invention can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, intranodal, andsubcutaneous for injection, the oligomers of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, theoligomers may be formulated in solid form and redissolved or suspendedimmediately prior to use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

EXAMPLES

1. Role of PRT3 in Virus-Like Particle (VLP) Budding

1. Objective:

Use RNAi to inhibit PRT3 gene expression and compare the efficiency ofviral budding and Gag expression and processing in treated and untreatedcells.

2. Study Plan:

HeLa SS-6 cells are transfected with mRNA-specific RNAi in order toknockdown the target proteins. Since maximal reduction of target proteinby RNAi is achieved after 48 hours, cells are transfected twice—first toreduce target mRNAs, and subsequently to express the viral Gag protein.The second transfection is performed with pNLenv (plasmid that encodesHIV) and with low amounts of RNAi to maintain the knockdown of targetprotein during the time of gag expression and budding of VLPs. Reductionin mRNA levels due to RNAi effect is verified by RT-PCR amplification oftarget mRNA.

3. Methods, Materials, Solutions

a. Methods

-   -   i. Transfections according to manufacturer's protocol and as        described in procedure.    -   ii. Protein determined by Bradford assay.    -   iii. SDS-PAGE in Hoeffer miniVE electrophoresis system. Transfer        in Bio-Rad mini-protean II wet transfer system. Blots visualized        using Typhoon system, and ImageQuant software (ABbiotech)

b. Materials Material Manufacturer Catalog # Batch # Lipofectamine 2000Life Technologies 11668-019 1112496 (LF2000) OptiMEM Life Technologies31985-047 3063119 RNAi Lamin A/C Self 13 RNAi TSG101 688 Self 65 RNAiPRT3 524 Self 81 plenvl1 PTAP Self 148 plenvl1 ATAP Self 149 Anti-p24polyclonal Seramun A-0236/5- antibody 10-01 Anti-Rabbit Cy5 Jackson144-175-115 48715 conjugated antibody 10% acrylamide Tris- LifeTechnologies NP0321 1081371 Glycine SDS-PAGE gel NitrocelluloseSchleicher & 401353 BA-83 membrane Schuell NuPAGE 20× transfer LifeTechnologies NP0006-1 224365 buffer 0.45 μm filter Schleicher & 10462100CS1018-1 Schuell

c. Solutions Compound Concentration Lysis Buffer Tris-HCl pH 7.6  50 mMMgCl₂  15 mM NaCl 150 mM Glycerol   10% EDTA  1 mM EGTA  1 mM ASB-14(add immediately    1% before use) 6 × Sample Tris-HCl, pH = 6.8 1MBuffer Glycerol   30% SDS   10% DTT  9.3% Bromophenol Blue 0.012% TBS-TTris pH = 7.6  20 mM NaCl 137 mM Tween-20  0.1%4. Procedure

a. Schedule Day 1 2 3 4 5 Plate Transfection I Passage Transfection IIExtract RNA cells (RNAi only) cells (RNAi and pNlenv) for RT-PCR (1:3)(12:00, PM) (post transfection) Extract RNA for Harvest VLPs RT-PCR andcells (pre-transfection)

b. Day 1

Plate HeLa SS-6 cells in 6-well plates (35 mm wells) at concentration of5×10⁵ cells/well.

c. Day 2

2 hours before transfection replace growth medium with 2 ml growthmedium without antibiotics.

Transfection I: RNAi A B [20 μM] OPtiMEM LF2000 mix Reaction RNAi nameTAGDA# Reactions RNAi [nM] μl (μl) (μl) 1 Lamin A/C 13 2 50 12.5 500 5002 Lamin A/C 13 1 50 6.25 250 250 3 TSG101 688 65 2 20 5 500 500 5 PRT3524 81 2 50 12.5 500 500Transfections:

Prepare LF2000 mix: 250 μl OptiMEM+5 μl LF2000 for each reaction. Mix byinversion, 5 times. Incubate 5 minutes at room temperature.

Prepare RNA dilution in OptiMEM (Table 1, column A). Add LF2000 mixdropwise to diluted RNA (Table 1, column B). Mix by gentle vortex.Incubate at room temperature 25 minutes, covered with aluminum foil.

Add 500 μl transfection mixture to cells dropwise and mix by rockingside to side. Incubate overnight.

d. Day 3

Split 1:3 after 24 hours. (Plate 4 wells for each reaction, exceptreaction 2 which is plated into 3 wells.)

e. Day 4

2 hours pre-transfection replace medium with DMEM growth medium withoutantibiotics.

Transfection II A B Plasmid RNAi [20 μM] C D Plasmid for 2.4 μg for 10nM OPtiMEM LF2000 mix Reaction RNAi name TAGDA# Plasmid Reactions(μg/μl) (μl) (μl) (μl) (μl) 1 Lamin A/C 13 PTAP 3 3.4 3.75 750 750 2Lamin A/C 13 ATAP 3 2.5 3.75 750 750 3 TSG101 688 65 PTAP 3 3.4 3.75 750750 5 PRT3 524 81 PTAP 3 3.4 3.75 750 750

Prepare LF2000 mix: 250 μl OptiMEM+5 μl LF2000 for each reaction. Mix byinversion, 5 times. Incubate 5 minutes at room temperature.

Prepare RNA+DNA diluted in OptiMEM (Transfection II, A+B+C)

Add LF2000 mix (Transfection II, D) to diluted RNA+DNA dropwise, mix bygentle vortex, and incubate 1 h while protected from light with aluminumfoil.

Add LF2000 and DNA+RNA to cells, 500 μl/well, mix by gentle rocking andincubate overnight.

f. Day 5

Collect samples for VLP assay (approximately 24 hours post-transfection)by the following procedure (cells from one well from each sample istaken for RNA assay, by RT-PCR).

g. Cell Extracts

-   -   i. Pellet floating cells by centrifugation (5 min, 3000 rpm at        40° C.), save supernatant (continue with supernatant immediately        to step h), scrape remaining cells in the medium which remains        in the well, add to the corresponding floating cell pellet and        centrifuge for 5 minutes, 1800 rpm at 40° C.    -   ii. Wash cell pellet twice with ice-cold PBS.    -   iii. Resuspend cell pellet in 100 μl lysis buffer and incubate        20 minutes on ice.    -   iv. Centrifuge at 14,000 rpm for 15 min. Transfer supernatant to        a clean tube. This is the cell extract.    -   v. Prepare 10 μl of cell extract samples for SDS-PAGE by adding        SDS-PAGE sample buffer to 1×, and boiling for 10 minutes. Remove        an aliquot of the remaining sample for protein determination to        verify total initial starting material. Save remaining cell        extract at −80° C.    -   h. Purification of VLPs from Cell Media    -   i. Filter the supernatant from step g through a 0.45 m filter.    -   ii. Centrifuge supernatant at 14,000 rpm at 40 C for at least        2h.    -   iii. Aspirate supernatant carefully.    -   iv. Re-suspend VLP pellet in hot (100° C. warmed for 10 min at        least) 1× sample buffer.    -   v. Boil samples for 10 minutes, 1001C.

i. Western Blot analysis

-   -   i. Run all samples from stages A and B on Tris-Glycine SDS-PAGE        10% (120V for 1.5 h.).    -   ii. Transfer samples to nitrocellulose membrane (65V for 1.5h.).    -   iii. Stain membrane with ponceau S solution.    -   iv. Block with 10% low fat milk in TBS-T for 1 h.    -   v. Incubate with anti p24 rabbit 1:500 in TBS-T o/n.    -   vi. Wash 3 times with TBS-T for 7 min each wash.    -   vii. Incubate with secondary antibody anti rabbit cy5 1:500 for        30 min.    -   viii. Wash five times for 10 min in TBS-T    -   ix. View in Typhoon gel imaging system (Molecular        Dynamics/APBiotech) for fluorescence signal.

Results are shown in FIGS. 82-84.

2. Exemplary PRT3 RT-PCR primers and siRNA duplexes

RT-PCR Primers Name Position Sequence Sense primer PRT3═271  271  5′CTTGCCTTGCCAGCATAC 3′ (SEQ ID NO:12) Anti-sense PRT3═926c 926C 5′CTGCCAGCATTCCTTCAG 3′ (SEQ ID NO:13) primer

siRNA Duplexes: siRNA No: 153 siRNA Name: PRT3-230 Position in mRNA426-446 Target sequence: 5′ AACAGAGGCCTTGGAAACCTG 3′ SEQ ID NO: 14 siRNAsense strand: 5′ dTdTCAGAGGCCUUGGAAACCUG 3′ SEQ ID NO: 15 siRNAanti-sense strand: 5′dTdTCAGGUUUCCAAGGCCUCUG 3′ SEQ ID NO: 16 siRNA No:155 siRNA Name: PRT3-442 Position in mRNA 638-658 Target sequence: 5′AAAGAGCCTGGAGACCTTAAA 3′ SEQ ID NO: 17 siRNA sense strand: 5′ddTdTAGAGCCUGGAGACCUUAAA 3′ SEQ ID NO: 18 siRNA anti-sense strand: 5′ddTdTUUUAAGGUCUCCAGGCUGU 3′ SEQ ID NO: 19 siRNA No: 157 siRNA Name:PRT3-U111 Position in mRNA 2973-2993 Target sequence: 5′AAGGATTGGTATGTGACTCTG 3′ SEQ ID NO: 20 siRNA sense strand: 5′dTdTGGAUUGGUAUGUGACUCUG 3′ SEQ ID NO: 21 siRNA anti-sense strand: 5′dTdTCAGAGUCACAUACCAAUCC 3′ SEQ ID NO: 22 siRNA No: 159 siRNA Name:PRT3-U410 Position in mRNA 3272-3292 Target sequence: 5′AAGCTGGATTATCTCCTGTTG 3′ SEQ ID NO: 23 siRNA sense strand: 5′ddTdTGCUGGAUUAUCUCCUGUUG 3′ SEQ ID NO: 24 siRNA anti-sense strand: 5′ddTdTCAACAGGAGAUAAUCCAGC 3′ SEQ ID NO: 253. Effects of PRT3 RNAi on HIV Release: Kinetics

A1. Transfections

-   -   1. One day before transfection plate cells at a concentration of        5×10⁶ cell/well in 15 cm plates.    -   2. Two hours before transfection, replace cell media to 20 ml        complete DMEM without antibiotics.    -   3. DNA dilution: for each transfection dilute 62.511 RNAi in 2.5        ml OptiMEM according to the table below. RNAi stock is 201M        (recommended concentration: 50 nM, dilution in total medium        amount 1:400).    -   4. LF 2000 dilution: for each transfection dilute 50 μl LF 2000        reagent in 2.5 ml OptiMEM.    -   5. Incubate diluted RNAi and LF 2000 for 5 minutes at RT.    -   6. Mix the diluted RNAi with diluted LF2000 and incubated for        20-25 minutes at RT.    -   7. Add the mixture to the cells (drop wise) and incubate for 24        hours at 37° C. in CO₂ incubator.    -   8. One day after RNAi transfection split cells (in complete MEM        medium to 2 15 cm plate and 1 well in a 6 wells plate)    -   9. One day after cell split perform HIV transfection.    -   10. 6 hours after HIV transfection replace medium to complete        MEM medium.    -   RT-PCR for PRT3 may be performed to assess the degree of        knockdown.

A2. Total RNA Purification.

-   -   1. One day after transfection, wash cells twice with sterile        PBS.

2. Scrape cells in 2.3 ml/200 μl (for 15 cm plate/1 well of a 6 wellsplate) Tri reagent (with sterile scrapers) and freeze in −70° C. Chasetime Treatment (hours) Fraction Labeling Control = WT 1 Cells A1 VLP A1V 2 Cells A2 VLP A2 V 3 Cells A3 VLP A3 V 4 Cells A4 VLP A4 V 5 Cells A5VLP A5 V PRT3 + WT 1 Cells B1 VLP B1 V 2 Cells B2 VLP B2 V 3 Cells B3VLP B3 V 4 Cells B4 VLP B4 V 5 Cells B5 VLP B5 V

B. Labeling

-   -   1. Take out starvation medium, thaw and place at 37° C.    -   2. Scrape cells in growth medium and transfer gently into 15 ml        conical tube.    -   3. Centrifuge to pellet cells at 1800 rpm for 5 minutes at room        temperature.    -   4. Aspirate supernatant and let tube stand for 10 sec. Remove        the rest of the supernatant with a 200%1 pipetman.    -   5. Gently add 10 ml warm starvation medium and resuspend        carefully with a 10 ml pipette, up and down, just turning may        not resolve the cell pellet).    -   6. Transfer cells to 10 cm tube and place in the incubator for        60 minutes. Set an Eppendorf thermo mixer to 37° C.    -   7. Centrifuge to pellet cells at 1800 rpm for 5 minutes at room        temperature.    -   8. Aspirate supernatant and let tube stand for 10 sec. Remove        the rest of the supernatant with a 200 μl pipetman.    -   9. Cut a 2001 μl tip from the end and resuspend cells (˜1.5 10⁷        cells in 150 μl RPIM without Met, but try not to go over 250 μl        if you have more cells) gently in 1501 μl starvation medium.        Transfer cells to an Eppendorf tube and place in the thermo        mixer. Wait 10 sec and transfer the rest of the cells from the        10 ml tube to the Eppendorf tube, if necessary add another 50 μl        to splash the rest of the cells out (all specimens should have        the same volume of labeling reaction).    -   10. Pulse: Add 50 μl of ³⁵S-methionine (specific activity 14.2        μCi/μl), tightly cup tubes and place in thermo mixer. Set the        mixing speed to the lowest possible (700 rpm) and incubate for        25 minutes.    -   11. Stop the pulse by adding 1 ml ice-cold chase/stop medium.        Shake tube very gently three times and pellet cells at 600 rpm        for 6 sec.    -   12. Remove supernatant with a 1 ml tip. Add gently 1 ml ice-cold        chase/stop medium to the pelleted cells and invert gently to        resuspend.    -   13. Chase: Transfer all tubes to the thermo mixer and incubate        for the required chase time (830: 1, 2, 3, 4 and 5 hours; 828: 3        hours only). At the end of total chase time, place tubes on ice,        add 1 ml ice-cold chase/stop and pellet cells for 1 minute at        14,000 rpm. Remove supernatant and transfer supernatant to a        second eppendorf tube. The cell pellet freeze at −80° C., until        all tubes are ready.    -   14. Centrifuge supernatants for 2 hours at 14,000 rpm, 4° C.        Remove the supernatant very gently, leave 20 μl in the tube        (labeled as V) and freeze at −80° C. until the end of the time        course.    -   *** All steps are done on ice with ice-cold buffers    -   15. When the time course is over, remove all tubes form −80° C.        Lyse VLP pellet (from step 14) and cell pellet (step 13) by        adding 500 μl of lysis buffer (see solutions), resuspend well by        pipeting up and down three times. Incubate on ice for 15        minutes, and spin in an eppendorf centrifuge for 15 minutes at        4° C., 14,000 rpm. Remove supernatant to a fresh tube, discard        pellet.

16. Perform IP with anti-p24 sheep for all samples.

C. Immunoprecipitation

-   -   1. Preclearing: add to all samples 15 μl ImmunoPure PlusG        (Pierce). Rotate for 1 hour at 4° C. in a cycler, spin 5 min at        4° C., and transfer to a new tube for IP.    -   2. Add to all samples 20 μl of p24-protein G conjugated beads        and incubate 4 hours in a cycler at 4° C.    -   3. Post immunoprecipitations, transfer all immunoprecipitations        to a fresh tube.    -   4. Wash beads once with high salt buffer, once with medium salt        buffer and once with low salt buffer. After each spin don't        remove all solution, but leave 50 μl solution on the beads.        After the last spin remove supernatant carefully with a loading        tip and leave ˜10 μl solution.    -   5. Add to each tube 20 μl 2×SDS sample buffer. Heat to 70° C.        for 10 minutes.    -   6. Samples were separated on 10% SDS-PAGE.    -   7. Fix gel in 25% ethanol and 10% acetic acid for 15 minutes.    -   8. Pour off the fixation solution and soak gels in Amplify        solution (NAMP 100 Amersham) for 15 minutes.    -   9. Dry gels on warm plate (60-80° C.) under vacuum.    -   10. Expose gels to screen for 2 hours and scan.        4. Prophetic Characterization of Viral Maturation Polypeptides        and Systems

The methods disclosed herein are useful for, among other things,identifying host proteins involved in virus release. To this end wecompare membrane protein profiles of cells infected with virusesexpressing wild type (wt) p6Gag (p6) with the parallel profiles fromcells infected with viruses harboring a mutant form of p6. Wt p6 isexpected to attract the release machinery and it is expected that themutant virus fails to do so. Furthermore, we also investigate themechanism of the exceedingly high efficiency release mechanism of theEbola virus by comparing the protein profiles of membranes from cellsinfected with mutant HIV-1 expressing the Ebola release determinant withmembranes of cells infected with wt virus.

Host Protein Profiling

Membranes from uninfected, wt virus and mutant virus-infected cells areprepared according to protocols modified to enable virus inactivationprior to sample handling and separation. The membranes samples areseparated by 2D gel electrophoresis (2DGE). The 2D maps are analyzed andproteins specific to wt virus are subjected to mass spectrometryanalysis.

Virion Protein Profiling

Host proteins involved in virus release are expected to be trapped invirions after their release. This expectation is based, in part, on twoobservations. The first is the presence of ubiquitin in virions atconcentrations higher than the ubiquitin cellular concentration. Thesecond is the finding that EIAV gag protein associates with AP50 andthat AP-2 is also found in EIAV particles. We have foundmono-ubiquitinated p9 of EIAV in the virions.

It is therefore possible that additional host proteins are included invirions. Analysis of host proteins included in virus particlesfacilitates identification of host proteins involved in virus buddingand release.

To identify host proteins included in virus particles, we harvest thevirions from the supernatant of virus-infected cells. The virions arethen by lysed and the proteins are subsequently analyzed by 2DGE.Specific antibodies are used to identify the viral proteins. Theunidentified host proteins are subjected to MS analysis. Antibodies tothe host proteins present in the virus particles are used to detect themin membranes of virus infected cells. It is believed that host proteinspresent in virions and localization in sites of virus budding will beinvolved in virus maturation.

Identification of Ubiquitinated Proteins Associated with Virus Release

Cells transfected with hemaglutinin (HA)-tagged ubiquitin are infectedwith the relevant virus. To isolate the ubiquitinated proteins,detergent lysates are prepared and the detergent extract is subjected toimmunoprecipitation with anti-HA antibody. The immunoprecipitates aresubjected to separation by either SDS-PAGE or 2DGE (depending on thecomplexity of the proteome). Using this approach we compareubiquitinated proteins from wt and mutant virus-infected cells. Thoseproteins that are ubiquitinated in wt but not in mutant-infected cellsare identified and characterized by mass spectrometry analysis.

Identification of Ubiquitin-Protein Ligases

The rate-limiting component for virus release is expected to be aubiquitin-protein ligase. The Ebola recruits the ligase to the sites ofbudding with exceedingly higher efficiency than any other retrovirus.

-   -   1. We generate a recombinant HIV1-p6 with the Ebola tandem L        motif. To confirm the Ebola potency we compare virus-like        particle release into the medium of cells expressing the two p6        forms. Virus-like particles are harvested from the medium of        p6-expressing cells. We quantitatively detect Gag with specific        antibodies by immunoblot analysis. It is expected that Gag        signal will be much higher in the supernatant of p6 Ebola        expressing cells.    -   2. We prepare membraned protein-enriched fraction. To this end        we prepare protein fractions from unwashed or mildly washed        membrane so as to minimally disturb possible ligase-protein        interactions. Protein profiles of plasma membrane proteins from        un-infected, p6 _(HIV-1) and P6 _(Ebola)-transfected cells are        compared. Proteins that are expressed at higher levels in the P6        Ebola membranes are analyzed and identified by mass        spectrometry. We specifically search for proteins with the        characteristics of an AVMSP.    -   3. Alternatively, we utilize anti-p6 antibodies to precipitate        proteins that are associated with p6. A 2D profile of        p6-associated proteinsis performed and results are analyzed via        a similar rational as described in part 2 above.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Equivalents

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. An isolated protein complex comprising a RING-SH₃ polypeptide incombination with at least one polypeptide selected from the groupconsisting of: a Gag protein, a Gag late domain, PI3K, actin, myosin,Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2enzyme, tsg101, a cullin, RING-SH₃, and a clathrin.
 2. The isolatedprotein complex of claim 1, wherein said Gag protein is an HIV gagprotein.
 3. The isolated protein complex of claim 1, wherein said Gagprotein comprises the Gag late domain.
 4. The isolated protein complexof claim 3, wherein said Gag late domain is PTAP.
 5. The isolatedprotein complex of claim 3, wherein said Gag late domain is PxxY.
 6. Theisolated protein complex of claim 3, wherein said Gag late domain isPxxL.
 7. The isolated protein complex of claim 3, wherein said Gag latedomain is PPxY.
 8. The isolated protein complex of claim 3, wherein saidGag late domain is YxxL.
 9. The isolated protein complex of claim 3,wherein said Gag late domain is PxxP.
 10. A host cell comprising a firstnucleic acid and a second nucleic acid, wherein the first nucleic acidcomprises a recombinant RING-SH₃ nucleic acid, and wherein the secondnucleic acid comprises a recombinant nucleic acid encoding a Gagprotein.
 11. The host cell of claim 10, wherein said Gag protein is anHIV gag protein.
 12. The host cell of claim 11, wherein said Gag proteincomprises the Gag late domain.
 13. The host cell of claim 12, whereinsaid Gag late domain is PTAP.
 14. The host cell of claim 12, whereinsaid Gag late domain is PxxY.
 15. The host cell of claim 12, whereinsaid Gag late domain is PxxL.
 16. The host cell of claim 12, whereinsaid Gag late domain is PPxY.
 17. The host cell of claim 12, whereinsaid Gag late domain is YxxL.
 18. The host cell of claim 12, whereinsaid Gag late domain is PxxP.
 19. A method for identifying modulators ofprotein complexes, comprising: (i) forming a reaction mixture comprising(a) a RING-SH₃; and (b) a second polypeptide selected from the groupconsisting of: RING-SH₃, a gag protein, a Gag late domain, PI3K, actin,myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase,an E2 enzyme, tsg101, a cullin, and a clathrin; (ii) contacting saidreaction mixture with a test agent, and (iii) determining the effect ofsaid test agent for one or more activities selected from the groupcomprising (a) a change in the level of the protein complex, (b) achange in the enzymatic activity of the complex, or (c) where thereaction mixture is a whole cell, a change in the plasma membranelocalization of the complex or a component thereof.
 20. A method foridentifying a test compound which inhibits or potentiates complexformation, comprising: (i) forming a reaction mixture comprising (a) aRING-SH₃; and (b) a second polypeptide selected from the groupconsisting of: RING-SH₃, a gag protein, a Gag late domain, P13K, actin,myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase,an E2 enzyme, tsg101, a cullin, and a clathrin; (ii) contacting saidreaction mixture with a test agent, and (iii) detecting binding of saidRING-SH₃ to said second polypeptide; wherein a change in the binding ofsaid RING-SH₃ to said second polypeptide in the presence of the testcompound, relative to binding in the absence of the test compound,indicates that said test compound potentiates or inhibits complexformation between said RING-SH₃ and said second polypeptide.
 21. Amethod for inhibiting infection in a subject in need thereof, comprisingadministering an effective amount of an agent that inhibits the bindingof a RING-SH₃ polypeptide to an gag protein.
 22. The method of claim 21,wherein said agent is selected from the group comprising a smallmolecule, a antibody, and a peptide.
 23. The method of claim 22, whereinthe Gag protein is an HIV Gag protein.
 24. The method of claim 22,wherein the Gag polypeptide is HIV p24.
 25. An isolated antibody, orfragment thereof, specifically immunoreactive with an epitope of aRING-SH₃ polypeptide, which disrupts the interaction between saidRING-SH₃ and a RING-SH₃-associating polypeptide (RING-SH₃-AP).
 26. Theantibody of claim 25, wherein said antibody is a monoclonal antibody.27. The antibody of claim 25, wherein said antibody is a Fab fragment.28. The antibody of claim 25, wherein said antibody is labeled with adetectable label.
 29. The antibody of claim 25, wherein said RING-SH₃-APis a gag polypeptide.
 30. The antibody of claim 25, wherein saidRING-SH₃-AP is an HIV gag polypeptide.
 31. A kit for detecting aRING-SH₃ polypeptide protein comprising (i) isolated anti-RING-SH₃antibodies, or fragment thereof, specifically immunoreactive with anepitope of an RING-SH₃, which epitope interacts with an RING-SH₃-AP, and(ii) a detectable label for detecting said anti-RING-SH₃ antibody inimmunoclomplexes with said RING-SH₃ polypeptide.
 32. A host cellcomprising a first nucleic acid and a second nucleic acid, wherein thefirst nucleic acid comprises a recombinant RING-SH₃ nucleic acid, andwherein the second nucleic acid comprises a recombinant nucleic acidencoding a HIV Gag protein.
 33. A method for inhibiting infection in asubject in need thereof, comprising administering an effective amount ofan agent that inhibits the binding of a RING-SH₃ polypeptide to an HIVgag protein.
 34. An anti-viral therapeutic composition comprising adouble stranded oligoribonucleotide molecule that inhibits expression ofa nucleic acid molecule encoding a RING-SH₃ polypeptide